COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF Eg5 GENE

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the Eg5 gene (Eg5 gene), comprising an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of the Eg5 gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by Eg5 expression and the expression of the Eg5 gene using the pharmaceutical composition; and methods for inhibiting the expression of the Eg5 gene in a cell.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/165,568, filed Jun. 21, 2011 (pending), which is a continuation ofU.S. application Ser. No. 12,754,110, filed Apr. 5, 2010 (abandoned),which is a divisional of U.S. application Ser. No. 11/694,215, filedMar. 30, 2007 (now U.S. Pat. No. 7,718,629) all which claim the benefitof U.S. Provisional Application No. 60/787,762, filed Mar. 31, 2006, andU.S. Provisional Application No. 60/870,259, filed Dec. 15, 2006. Allprior applications are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), andits use in mediating RNA interference to inhibit the expression of theEg5 gene and the use of the dsRNA to treat pathological processesmediated by Eg5 expression, such as cancer, alone or in combination witha dsRNA targeting vascular endothelian growth factor (VEGF).

BACKGROUND OF THE INVENTION

The maintenance of cell populations within an organism is governed bythe cellular processes of cell division and programmed cell death.Within normal cells, the cellular events associated with the initiationand completion of each process is highly regulated. In proliferativedisease such as cancer, one or both of these processes may be perturbed.For example, a cancer cell may have lost its regulation (checkpointcontrol) of the cell division cycle through either the overexpression ofa positive regulator or the loss of a negative regulator, perhaps bymutation.

Alternatively, a cancer cell may have lost the ability to undergoprogrammed cell death through the overexpression of a negativeregulator. Hence, there is a need to develop new chemotherapeutic drugsthat will restore the processes of checkpoint control and programmedcell death to cancerous cells.

One approach to the treatment of human cancers is to target a proteinthat is essential for cell cycle progression. In order for the cellcycle to proceed from one phase to the next, certain prerequisite eventsmust be completed. There are checkpoints within the cell cycle thatenforce the proper order of events and phases. One such checkpoint isthe spindle checkpoint that occurs during the metaphase stage ofmitosis. Small molecules that target proteins with essential functionsin mitosis may initiate the spindle checkpoint to arrest cells inmitosis. Of the small molecules that arrest cells in mitosis, thosewhich display anti-tumor activity in the clinic also induce apoptosis,the morphological changes associated with programmed cell death. Aneffective chemotherapeutic for the treatment of cancer may thus be onewhich induces checkpoint control and programmed cell death.Unfortunately, there are few compounds available for controlling theseprocesses within the cell. Most compounds known to cause mitotic arrestand apoptosis act as tubulin binding agents. These compounds alter thedynamic instability of microtubules and indirectly alter thefunction/structure of the mitotic spindle thereby causing mitoticarrest. Because most of these compounds specifically target the tubulinprotein which is a component of all microtubules, they may also affectone or more of the numerous normal cellular processes in whichmicrotubules have a role. Hence, there is also a need for smallmolecules that more specifically target proteins associated withproliferating cells.

Eg5 is one of several kinesin-like motor proteins that are localized tothe mitotic spindle and known to be required for formation and/orfunction of the bipolar mitotic spindle. Recently, there was a report ofa small molecule that disturbs bipolarity of the mitotic spindle (Mayer,T. U. et. al. 1999. Science 286(5441) 971-4, herein incorporated byreference). More specifically, the small molecule induced the formationof an aberrant mitotic spindle wherein a monoastral array ofmicrotubules emanated from a central pair of centrosomes, withchromosomes attached to the distal ends of the microtubules. The smallmolecule was dubbed “monastrol” after the monoastral array. Thismonoastral array phenotype had been previously observed in mitotic cellsthat were immunodepleted of the Eg5 motor protein. This distinctivemonoastral array phenotype facilitated identification of monastrol as apotential inhibitor of Eg5. Indeed, monastrol was further shown toinhibit the Eg5 motor-driven motility of microtubules in an in vitroassay. The Eg5 inhibitor monastrol had no apparent effect upon therelated kinesin motor or upon the motor(s) responsible for golgiapparatus movement within the cell. Cells that display the monoastralarray phenotype either through immunodepletion of Eg5 or monastrolinhibition of Eg5 arrest in M-phase of the cell cycle. However, themitotic arrest induced by either immunodepletion or inhibition of Eg5 istransient (Kapoor, T. M., 2000. J Cell Biol 150(5) 975-80). Both themonoastral array phenotype and the cell cycle arrest in mitosis inducedby monastrol are reversible. Cells recover to form a normal bipolarmitotic spindle, to complete mitosis and to proceed through the cellcycle and normal cell proliferation. These data suggest that a smallmolecule inhibitor of Eg5 which induced a transient mitotic arrest maynot be effective for the treatment of cancer cell proliferation.Nonetheless, the discovery that monastrol causes mitotic arrest isintriguing and hence there is a need to further study and identifycompounds which can be used to modulate the Eg5 motor protein in amanner that would be effective in the treatment of human cancers. Thereis also a need to explore the use of these compounds in combination withother antineoplastic agents.

VEGF (also known as vascular permeability factor, VPF) is amultifunctional cytokine that stimulates angiogenesis, epithelial cellproliferation, and endothelial cell survival. VEGF can be produced by awide variety of tissues, and its overexpression or aberrant expressioncan result in a variety disorders, including cancers and retinaldisorders such as age-related macular degeneration and other angiogenicdisorders.

Recently, double-stranded RNA molecules (dsRNA) have been shown to blockgene expression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant or unwanted regulation of a gene.

Despite significant advances in the field of RNAi and advances in thetreatment of pathological processes mediated by Eg5 expression, thereremains a need for an agent that can selectively and efficiently silencethe Eg5 gene using the cell's own RNAi machinery that has both highbiological activity and in vivo stability, and that can effectivelyinhibit expression of a target Eg5 gene for use in treating pathologicalprocesses mediated by Eg5 expression.

SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the Eg5gene in a cell or mammal using such dsRNA, alone or in combination witha dsRNA targeting VEGF. The invention also provides compositions andmethods for treating pathological conditions and diseases caused by theexpression of the Eg5 gene, such as in cancer. The dsRNA of theinvention comprises an RNA strand (the antisense strand) having a regionwhich is less than 30 nucleotides in length, generally 19-24 nucleotidesin length, and is substantially complementary to at least part of anmRNA transcript of the Eg5 gene.

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the Eg5 gene.The dsRNA comprises at least two sequences that are complementary toeach other. The dsRNA comprises a sense strand comprising a firstsequence and an antisense strand comprising a second sequence. Theantisense strand comprises a nucleotide sequence which is substantiallycomplementary to at least part of an mRNA encoding Eg5, and the regionof complementarity is less than 30 nucleotides in length, generally19-24 nucleotides in length. The dsRNA, upon contacting with a cellexpressing the Eg5, inhibits the expression of the Eg5 gene by at least40%.

For example, the dsRNA molecules of the invention can be comprised of afirst sequence of the dsRNA that is selected from the group consistingof the sense sequences of Tables 1-3 and the second sequence is selectedfrom the group consisting of the antisense sequences of Tables 1-3. ThedsRNA molecules of the invention can be comprised of naturally occurringnucleotides or can be comprised of at least one modified nucleotide,such as a 2′-O-methyl modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative. Alternatively, the modified nucleotide may bechosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholinonucleotide, a phosphoramidate, and a non-natural base comprisingnucleotide. Generally, such modified sequence will be based on a firstsequence of said dsRNA selected from the group consisting of the sensesequences of Tables 1-3 and a second sequence selected from the groupconsisting of the antisense sequences of Tables 1-3.

In another embodiment, the invention provides a cell comprising one ofthe dsRNAs of the invention. The cell is generally a mammalian cell,such as a human cell.

In another embodiment, the invention provides a pharmaceuticalcomposition for inhibiting the expression of the Eg5 gene in anorganism, generally a human subject, comprising one or more of the dsRNAof the invention and a pharmaceutically acceptable carrier or deliveryvehicle.

In another embodiment, the invention provides a method for inhibitingthe expression of the Eg5 gene in a cell, comprising the followingsteps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA comprises at least two sequences that        are complementary to each other. The dsRNA comprises a sense        strand comprising a first sequence and an antisense strand        comprising a second sequence. The antisense strand comprises a        region of complementarity which is substantially complementary        to at least a part of a mRNA encoding Eg5, and wherein the        region of complementarity is less than 30 nucleotides in length,        generally 19-24 nucleotides in length, and wherein the dsRNA,        upon contact with a cell expressing the Eg5, inhibits expression        of the Eg5 gene by at least 40%; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of the        Eg5 gene, thereby inhibiting expression of the Eg5 gene in the        cell.

In another embodiment, the invention provides methods for treating,preventing or managing pathological processes mediated by Eg5expression, e.g. cancer, comprising administering to a patient in needof such treatment, prevention or management a therapeutically orprophylactically effective amount of one or more of the dsRNAs of theinvention.

In another embodiment, the invention provides vectors for inhibiting theexpression of the Eg5 gene in a cell, comprising a regulatory sequenceoperably linked to a nucleotide sequence that encodes at least onestrand of one of the dsRNA of the invention.

In another embodiment, the invention provides a cell comprising a vectorfor inhibiting the expression of the Eg5 gene in a cell. The vectorcomprises a regulatory sequence operably linked to a nucleotide sequencethat encodes at least one strand of one of the dsRNA of the invention.

In a further embodiment, the invention provides the Eg5 dsRNA and theuses thereof as described above in combination with a second dsRNAtargeting the VEGF mRNA. A combination of a dsRNA targeting Eg5 and asecond dsRNA targeting VEGF provides complementary and synergiaticactivity for treating hyperproliferative discords, particularly hepaticcarcinoma.

BRIEF DESCRIPTION OF THE FIGURES

No Figures are presented

DETAILED DESCRIPTION OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the Eg5gene in a cell or mammal using the dsRNA. The invention also providescompositions and methods for treating pathological conditions anddiseases in a mammal caused by the expression of the Eg5 gene usingdsRNA. dsRNA directs the sequence-specific degradation of mRNA through aprocess known as RNA interference (RNAi). The invention further providesthis dsRNA in combination with a second dsRNA that inhibits theexpression of the VEGF gene.

The dsRNAs of the invention comprises an RNA strand (the antisensestrand) having a region which is less than 30 nucleotides in length,generally 19-24 nucleotides in length, and is substantiallycomplementary to at least part of an mRNA transcript of the Eg5 gene.The use of these dsRNAs enables the targeted degradation of mRNAs ofgenes that are implicated in replication and or maintenance of cancercells in mammals. Using cell-based and animal assays, the presentinventors have demonstrated that very low dosages of these dsRNA canspecifically and efficiently mediate RNAi, resulting in significantinhibition of expression of the Eg5 gene. Thus, the methods andcompositions of the invention comprising these dsRNAs are useful fortreating pathological processes mediated by Eg5 expression, e.g. cancer,by targeting a gene involved in mitotic division.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression of theEg5 gene, as well as compositions and methods for treating diseases anddisorders caused by the expression of Eg5, such as cancer, alone or incombination with a second dsRNA targeting the VEGF gene. Thepharmaceutical compositions of the invention comprise a dsRNA having anantisense strand comprising a region of complementarity which is lessthan 30 nucleotides in length, generally 19-24 nucleotides in length,and is substantially complementary to at least part of an RNA transcriptof the Eg5 gene, together with a pharmaceutically acceptable carrier. Asdiscussed above, such compositions can further include a second dsRNAtargeting VEGF.

Accordingly, certain aspects of the invention provide pharmaceuticalcompositions comprising the dsRNA of the invention together with apharmaceutically acceptable carrier, methods of using the compositionsto inhibit expression of the Eg5 gene, and methods of using thepharmaceutical compositions to treat diseases caused by expression ofthe Eg5 gene. The invention further provides the above pharmaceuticalcompositions further containing a second dsRNA designed to inhibit theexpression of VEGF.

I. DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.However, it will be understood that the term “ribonucleotide” or“nucleotide” can also refer to a modified nucleotide, as furtherdetailed below, or a surrogate replacement moiety. The skilled person iswell aware that guanine, cytosine, adenine, and uracil may be replacedby other moieties without substantially altering the base pairingproperties of an oligonucleotide comprising a nucleotide bearing suchreplacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of theinvention by a nucleotide containing, for example, inosine. Sequencescomprising such replacement moieties are embodiments of the invention.

As used herein, “Eg5” refers to the human kinesin family member 11,which is also known as KIF11, Eg5, HKSP, KNSL1 or TRIP5. Eg5 sequencecan be found as NCBI GeneID:3832, HGNC ID: HGNC:6388 and RefSeq IDnumber:NM_(—)004523.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof the Eg5 gene, including mRNA that is a product of RNA processing of aprimary transcription product.

As used herein, VEGF, also known as vascular permeability factor, is anangiogenic growth factor. VEGF is a homodimeric 45 kDa glycoprotein thatexists in at least three different isoforms. VEGF isoforms are expressedin endothelial cells. The VEGF gene contains 8 exons that express a189-amino acid protein isoform. A 165-amino acid isoform lacks theresidues encoded by exon 6, whereas a 121-amino acid isoform lacks theresidues encoded by exons 6 and 7. VEGF145 is an isoform predicted tocontain 145 amino acids and to lack exon 7. VEGF can act on endothelialcells by binding to an endothelial tyrosine kinase receptor, such asFlt-1 (VEGFR-1) or KDR/flk-1 (VEGFR-2). VEGFR-2 is expressed inendothelial cells and is involved in endothelial cell differentiationand vasculogenesis. A third receptor, VEGFR-3 has been implicated inlymphogenesis.

The various isoforms have different biologic activities and clinicalimplications. For example, VEGF145 induces angiogenesis and like VEGF189(but unlike VEGF165) VEGF145 binds efficiently to the extracellularmatrix by a mechanism that is not dependent on extracellularmatrix-associated heparin sulfates. VEGF displays activity as anendothelial cell mitogen and chemoattractant in vitro and inducesvascular permeability and angiogenesis in vivo. VEGF is secreted by awide variety of cancer cell types and promotes the growth of tumors byinducing the development of tumor-associated vasculature. Inhibition ofVEGF function has been shown to limit both the growth of primaryexperimental tumors as well as the incidence of metastases inimmunocompromised mice. Various dsRNAs directed to VEGF are described inco-pending U.S. Ser. No. 11/078,073 and Ser. No. 11/340,080, hereinincorporated by reference).

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidewhich is substantially complementary to a contiguous portion of the mRNAof interest (e.g., encoding Eg5). For example, a polynucleotide iscomplementary to at least a part of a Eg5 mRNA if the sequence issubstantially complementary to a non-interrupted portion of a mRNAencoding Eg5.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary, as definedabove, nucleic acid strands. The two strands forming the duplexstructure may be different portions of one larger RNA molecule, or theymay be separate RNA molecules. Where the two strands are part of onelarger molecule, and therefore are connected by an uninterrupted chainof nucleotides between the 3′-end of one strand and the 5′ end of therespective other strand forming the duplex structure, the connecting RNAchain is referred to as a “hairpin loop”. Where the two strands areconnected covalently by means other than an uninterrupted chain ofnucleotides between the 3′-end of one strand and the 5′ end of therespective other strand forming the duplex structure, the connectingstructure is referred to as a “linker”. The RNA strands may have thesame or a different number of nucleotides. The maximum number of basepairs is the number of nucleotides in the shortest strand of the dsRNAminus any overhangs that are present in the duplex. In addition to theduplex structure, a dsRNA may comprise one or more nucleotide overhangs.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

“Introducing into a cell”, when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell”, wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence” and “inhibit the expression of”, in as far as theyrefer to the Eg5 gene, herein refer to the at least partial suppressionof the expression of the Eg5 gene, as manifested by a reduction of theamount of mRNA transcribed from the Eg5 gene which may be isolated froma first cell or group of cells in which the Eg5 gene is transcribed andwhich has or have been treated such that the expression of the Eg5 geneis inhibited, as compared to a second cell or group of cellssubstantially identical to the first cell or group of cells but whichhas or have not been so treated (control cells). The degree ofinhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to Eg5 genetranscription, e.g. the amount of protein encoded by the Eg5 gene whichis secreted by a cell, or the number of cells displaying a certainphenotype, e.g apoptosis. In principle, Eg5 gene silencing may bedetermined in any cell expressing the target, either constitutively orby genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given dsRNA inhibitsthe expression of the Eg5 gene by a certain degree and therefore isencompassed by the instant invention, the assay provided in the Examplesbelow shall serve as such reference.

For example, in certain instances, expression of the Eg5 gene (or VEGFgene) is suppressed by at least about 20%, 25%, 35%, or 50% byadministration of the double-stranded oligonucleotide of the invention.In some embodiment, the Eg5 gene is suppressed by at least about 60%,70%, or 80% by administration of the double-stranded oligonucleotide ofthe invention. In some embodiments, the Eg5 gene is suppressed by atleast about 85%, 90%, or 95% by administration of the double-strandedoligonucleotide of the invention. Tables 1-3 provides values forinhibition of expression using various Eg5 dsRNA molecules at variousconcentrations.

As used herein in the context of Eg5 expression, the terms “treat”,“treatment”, and the like, refer to relief from or alleviation ofpathological processes mediated by Eg5 expression. In the context of thepresent invention insofar as it relates to any of the other conditionsrecited herein below (other than pathological processes mediated by Eg5expression), the terms “treat”, “treatment”, and the like mean torelieve or alleviate at least one symptom associated with suchcondition, or to slow or reverse the progression of such condition, suchas the slowing and progression of hepatic carcinoma.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes mediated by Eg5 expression or an overt symptom ofpathological processes mediated by Eg5 expression (alone or incombination with VEGF expression). The specific amount that istherapeutically effective can be readily determined by ordinary medicalpractitioner, and may vary depending on factors known in the art, suchas, e.g. the type of pathological processes mediated by Eg5 expression,the patient's history and age, the stage of pathological processesmediated by Eg5 expression, and the administration of otheranti-pathological processes mediated by Eg5 expression agents.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

II. DOUBLE-STRANDED RIBONUCLEIC ACID (DSRNA)

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the Eg5 gene(alone or incombinaton with a second dsRNA for inhibiting the expressionof VEGF) in a cell or mammal, wherein the dsRNA comprises an antisensestrand comprising a region of complementarity which is complementary toat least a part of an mRNA formed in the expression of the Eg5 gene, andwherein the region of complementarity is less than 30 nucleotides inlength, generally 19-24 nucleotides in length, and wherein said dsRNA,upon contact with a cell expressing said Eg5 gene, inhibits theexpression of said Eg5 gene by at least 40%. The dsRNA comprises two RNAstrands that are sufficiently complementary to hybridize to form aduplex structure. One strand of the dsRNA (the antisense strand)comprises a region of complementarity that is substantiallycomplementary, and generally fully complementary, to a target sequence,derived from the sequence of an mRNA formed during the expression of theEg5 gene, the other strand (the sense strand) comprises a region whichis complementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. Generally, the duplex structure is between 15 and 30, moregenerally between 18 and 25, yet more generally between 19 and 24, andmost generally between 19 and 21 base pairs in length. Similarly, theregion of complementarity to the target sequence is between 15 and 30,more generally between 18 and 25, yet more generally between 19 and 24,and most generally between 19 and 21 nucleotides in length. The dsRNA ofthe invention may further comprise one or more single-strandednucleotide overhang(s). The dsRNA can be synthesized by standard methodsknown in the art as further discussed below, e.g., by use of anautomated DNA synthesizer, such as are commercially available from, forexample, Biosearch, Applied Biosystems, Inc. In a preferred embodiment,the Eg5 gene is the human Eg5 gene. In specific embodiments, theantisense strand of the dsRNA comprises the sense sequences of Tables1-3 and the second sequence is selected from the group consisting of theantisense sequences of Tables 1-3. Alternative antisense agents thattarget elsewhere in the target sequence provided in Tables 1-3 canreadily be determined using the target sequence and the flanking Eg5sequence. In embodiments using a second dsRNA targeting VEGF, suchagents are exemplified in the Examples and in co-pending U.S. Ser. Nos.11/078,073 and 11/340,080, herein incorporated by reference.

The dsRNA will comprise at least two nucleotide sequence selected fromthe groups of sequences provided in Tables 1-3. One of the two sequencesis complementary to the other of the two sequences, with one of thesequences being substantially complementary to a sequence of an mRNAgenerated in the expression of the Eg5 gene. As such, the dsRNA willcomprises two oligonucleotides, wherein one oligonucleotide is describedas the sense strand in Tables 1-3 and the second oligonucleotide isdescribed as the antisense strand in Tables 1-3

The skilled person is well aware that dsRNAs comprising a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen hailed as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Tables 1-3, the dsRNAs of theinvention can comprise at least one strand of a length of minimally 21nt. It can be reasonably expected that shorter dsRNAs comprising one ofthe sequences of Tables 1-3 minus only a few nucleotides on one or bothends may be similarly effective as compared to the dsRNAs describedabove. Hence, dsRNAs comprising a partial sequence of at least 15, 16,17, 18, 19, 20, or more contiguous nucleotides from one of the sequencesof Tables 1-3, and differing in their ability to inhibit the expressionof the Eg5 gene in a FACS assay as described herein below by not morethan 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising thefull sequence, are contemplated by the invention. Further dsRNAs thatcleave within the target sequence provided in Tables 1-3 can readily bemade using the Eg5 sequence and the target sequence provided.

In addition, the RNAi agents provided in Tables 1-3 identify a site inthe Eg5 mRNA that is susceptible to RNAi based cleavage. As such thepresent invention further includes RNAi agents that target within thesequence targeted by one of the agents of the present invention. As usedherein a second RNAi agent is said to target within the sequence of afirst RNAi agent if the second RNAi agent cleaves the message anywherewithin the mRNA that is complementary to the antisense strand of thefirst RNAi agent. Such a second agent will generally consist of at least15 contiguous nucleotides from one of the sequences provided in Tables1-3 coupled to additional nucleotide sequences taken from the regioncontiguous to the selected sequence in the Eg5 gene. For example, thelast 15 nucleotides of SEQ ID NO:1 combined with the next 6 nucleotidesfrom the target Eg5 gene produces a single strand agent of 21nucleotides that is based on one of the sequences provided in Tables1-3.

The dsRNA of the invention can contain one or more mismatches to thetarget sequence. In a preferred embodiment, the dsRNA of the inventioncontains no more than 3 mismatches. If the antisense strand of the dsRNAcontains mismatches to a target sequence, it is preferable that the areaof mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of the Eg5 gene, the dsRNA generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed within the invention can be used to determine whether a dsRNAcontaining a mismatch to a target sequence is effective in inhibitingthe expression of the Eg5 gene. Consideration of the efficacy of dsRNAswith mismatches in inhibiting expression of the Eg5 gene is important,especially if the particular region of complementarity in the Eg5 geneis known to have polymorphic sequence variation within the population.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties than their blunt-ended counterparts. Moreover, thepresent inventors have discovered that the presence of only onenucleotide overhang strengthens the interference activity of the dsRNA,without affecting its overall stability. dsRNA having only one overhanghas proven particularly stable and effective in vivo, as well as in avariety of cells, cell culture mediums, blood, and serum. Generally, thesingle-stranded overhang is located at the 3′-terminal end of theantisense strand or, alternatively, at the 3′-terminal end of the sensestrand. The dsRNA may also have a blunt end, generally located at the5′-end of the antisense strand. Such dsRNAs have improved stability andinhibitory activity, thus allowing administration at low dosages, i.e.,less than 5 mg/kg body weight of the recipient per day. Generally, theantisense strand of the dsRNA has a nucleotide overhang at the 3′-end,and the 5′-end is blunt. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids of the invention may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Specific examples of preferred dsRNAcompounds useful in this invention include dsRNAs containing modifiedbackbones or no natural internucleoside linkages. As defined in thisspecification, dsRNAs having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified dsRNAs that do not havea phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified dsRNA backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Preferred modified dsRNA backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatoms and alkyl orcycloalkyl internucleoside linkages, or ore or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other preferred dsRNA mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an dsRNA mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar backbone of an dsRNA is replacedwith an amide containing backbone, in particular an aminoethylglycinebackbone. The nucleobases are retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found in Nielsen et al.,Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are dsRNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known asa methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Preferred dsRNAs comprise one of the following at the 2′ position: OH;F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C.sub.1 to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred dsRNAs comprise one of the following at the 2′ position:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃,SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an dsRNA, or a group for improving thepharmacodynamic properties of an dsRNA, and other substituents havingsimilar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Hely. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxygroup. A further preferred modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-OCH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro(2′-F). Similarmodifications may also be made at other positions on the dsRNA,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide.DsRNAs may also have sugar mimetics such as cyclobutyl moieties in placeof the pentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

DsRNAs may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., DsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Another modification of the dsRNAs of the invention involves chemicallylinking to the dsRNA one or more moieties or conjugates which enhancethe activity, cellular distribution or cellular uptake of the dsRNA.Such moieties include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199,86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let.,1994 4 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan etal., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg.Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNAconjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporatedby reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an dsRNA. The present invention also includesdsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compoundsor “chimeras,” in the context of this invention, are dsRNA compounds,particularly dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of an dsRNA compound. These dsRNAs typically contain atleast one region wherein the dsRNA is modified so as to confer upon thedsRNA increased resistance to nuclease degradation, increased cellularuptake, and/or increased binding affinity for the target nucleic acid.An additional region of the dsRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of anRNA:DNAduplex. Activation of RNase H, therefore, results in cleavage ofthe RNA target, thereby greatly enhancing the efficiency of dsRNAinhibition of gene expression. Consequently, comparable results canoften be obtained with shorter dsRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxydsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to dsRNAs in orderto enhance the activity, cellular distribution or cellular uptake of thedsRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such dsRNA conjugates have been listed above.Typical conjugation protocols involve the synthesis of dsRNAs bearing anaminolinker at one or more positions of the sequence. The amino group isthen reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction may beperformed either with the dsRNA still bound to the solid support orfollowing cleavage of the dsRNA in solution phase. Purification of thedsRNA conjugate by HPLC typically affords the pure conjugate.

Vector Encoded RNAi Agents

The dsRNA of the invention can also be expressed from recombinant viralvectors intracellularly in vivo. The recombinant viral vectors of theinvention comprise sequences encoding the dsRNA of the invention and anysuitable promoter for expressing the dsRNA sequences. Suitable promotersinclude, for example, the U6 or H1 RNA pol III promoter sequences andthe cytomegalovirus promoter. Selection of other suitable promoters iswithin the skill in the art. The recombinant viral vectors of theinvention can also comprise inducible or regulatable promoters forexpression of the dsRNA in a particular tissue or in a particularintracellular environment. The use of recombinant viral vectors todeliver dsRNA of the invention to cells in vivo is discussed in moredetail below.

dsRNA of the invention can be expressed from a recombinant viral vectoreither as two separate, complementary RNA molecules, or as a single RNAmolecule with two complementary regions.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorswhich express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801,the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the dsRNA of the invention isexpressed as two separate, complementary single-stranded RNA moleculesfrom a recombinant AAV vector comprising, for example, either the U6 orH1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA of the invention, a methodfor constructing the recombinant AV vector, and a method for deliveringthe vector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA of the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent ApplicationNo. WO 94/13788; and International Patent Application No. WO 93/24641,the entire disclosures of which are herein incorporated by reference.

III. PHARMACEUTICAL COMPOSITIONS COMPRISING DSRNA

In one embodiment, the invention provides pharmaceutical compositionscomprising a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition comprising the dsRNAis useful for treating a disease or disorder associated with theexpression or activity of the Eg5 gene, such as pathological processesmediated by Eg5 expression. Such pharmaceutical compositions areformulated based on the mode of delivery. One example is compositionsthat are formulated for systemic administration via parenteral delivery.

In another embodiment, such compositions will further comprise a seconddsRNA that inhibits VEGF expression. dsRNA directed to VEGF aredescribed in the Examples and in co-pending U.S. Ser. Nos. 11/078,073and 11/340,080.

The pharmaceutical compositions of the invention are administered indosages sufficient to inhibit expression of the Eg5 gene (and VEGFexpression when a second dsRNA is included). In general, a suitable doseof dsRNA will be in the range of 0.01 to 5.0 milligrams per kilogrambody weight of the recipient per day, generally in the range of 1microgram to 1 mg per kilogram body weight per day. The pharmaceuticalcomposition may be administered once daily or the dsRNA may beadministered as two, three, or more sub-doses at appropriate intervalsthroughout the day or even using continuous infusion or delivery througha controlled release formulation. In that case, the dsRNA contained ineach sub-dose must be correspondingly smaller in order to achieve thetotal daily dosage. The dosage unit can also be compounded for deliveryover several days, e.g., using a conventional sustained releaseformulation which provides sustained release of the dsRNA over a severalday period. Sustained release formulations are well known in the art andare particularly useful for delivery of agents at a particular site,such as could be used with the agents of the present invention. In thisembodiment, the dosage unit contains a corresponding multiple of thedaily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by Eg5 expression. Such models are used for in vivo testing ofdsRNA, as well as for determining a therapeutically effective dose.

The present invention also includes pharmaceutical compositions andformulations which include the dsRNA compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical, pulmonary, e.g., by inhalation or insufflation of powders oraerosols, including by nebulizer; intratracheal, intranasal, epidermaland transdermal), oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical formulations include those inwhich the dsRNAs of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). DsRNAs of the invention may be encapsulated withinliposomes or may form complexes thereto, in particular to cationicliposomes. Alternatively, dsRNAs may be complexed to lipids, inparticular to cationic lipids. Preferred fatty acids and esters includebut are not limited arachidonic acid, oleic acid, eicosanoic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which dsRNAs of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferredfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also preferred are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly preferred combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsof the invention may be delivered orally, in granular form includingsprayed dried particles, or complexed to form micro or nanoparticles.DsRNA complexing agents include poly-amino acids; polyimines;polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S.application. Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999), each of which is incorporated herein by referencein their entirety.

Compositions and formulations for parenteral, intrathecal,intraventricular or intrahepatic administration may include sterileaqueous solutions which may also contain buffers, diluents and othersuitable additives such as, but not limited to, penetration enhancers,carrier compounds and other pharmaceutically acceptable carriers orexcipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1.mu.min diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising two immiscible liquid phases intimately mixed and dispersedwith each other. In general, emulsions may be of either the water-in-oil(w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finelydivided into and dispersed as minute droplets into a bulk oily phase,the resulting composition is called a water-in-oil (w/o) emulsion.Alternatively, when an oily phase is finely divided into and dispersedas minute droplets into a bulk aqueous phase, the resulting compositionis called an oil-in-water (o/w) emulsion. Emulsions may containadditional components in addition to the dispersed phases, and theactive drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of ease of formulation, as well as efficacyfrom an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions of dsRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C 10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or dsRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of dsRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofdsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the dsRNAs and nucleicacids of the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and Novasome™II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether)were used to deliver cyclosporin-A into the dermis of mouse skin.Results indicated that such non-ionic liposomal systems were effectivein facilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G.sub.M1, or (B)is derivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G.sub.M1,galactocerebroside sulfate and phosphatidylinositol to improve bloodhalf-lives of liposomes. These findings were expounded upon by Gabizonet al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No.4,837,028 and WO 88/04924, both to Allen et al., disclose liposomescomprising (1) sphingomyelin and (2) the ganglioside G.sub.M1 or agalactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.)discloses liposomes comprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C.sub.1215G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an dsRNA RNA.U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods ofencapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love etal. discloses liposomes comprising dsRNA dsRNAs targeted to the rafgene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly dsRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of dsRNAs through the mucosa isenhanced. In addition to bile salts and fatty acids, these penetrationenhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyland t-butyl), and mono- and di-glycerides thereof (i.e., oleate,laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Leeet al., Critical Reviews in Therapeutic Drug Carryier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44,651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of dsRNAs through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of dsRNAs throughthe alimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of dsRNAs at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited todaunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosinearabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphor-amide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulation a range of dosage for use in humans. The dosage ofcompositions of the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, the dsRNAs of the invention can be administered incombination with other known agents effective in treatment ofpathological processes mediated by Eg5 expression. In any event, theadministering physician can adjust the amount and timing of dsRNAadministration on the basis of results observed using standard measuresof efficacy known in the art or described herein.

Methods for Treating Diseases Caused by Expression of the Eg5 Gene

The invention relates in particular to the use of a dsRNA or apharmaceutical composition prepared therefrom for the treatment ofcancer, e.g., for inhibiting tumor growth and tumor metastasis. Forexample, the dsRNA or a pharmaceutical composition prepared therefrommay be used for the treatment of solid tumors, like breast cancer, lungcancer, head and neck cancer, brain cancer, abdominal cancer, coloncancer, colorectal cancer, esophagus cancer, gastrointestinal cancer,glioma, liver cancer, tongue cancer, neuroblastoma, osteosarcoma,ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma,Wilm's tumor, multiple myeloma and for the treatment of skin cancer,like melanoma, for the treatment of lymphomas and blood cancer. Theinvention further relates to the use of an dsRNA according to theinvention or a pharmaceutical composition prepared therefrom forinhibiting eg5 expression and/or for inhibiting accumulation of ascitesfluid and pleural effusion in different types of cancer, e.g., breastcancer, lung cancer, head cancer, neck cancer, brain cancer, abdominalcancer, colon cancer, colorectal cancer, esophagus cancer,gastrointestinal cancer, glioma, liver cancer, tongue cancer,neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, retinoblastoma, Wilm's tumor, multiple myeloma, skin cancer,melanoma, lymphomas and blood cancer. Owing to the inhibitory effect oneg5 expression, an dsRNA according to the invention or a pharmaceuticalcomposition prepared therefrom can enhance the quality of life.

The invention furthermore relates to the use of an dsRNA or apharmaceutical composition thereof, e.g., for treating cancer or forpreventing tumor metastasis, in combination with other pharmaceuticalsand/or other therapeutic methods, e.g., with known pharmaceuticalsand/or known therapeutic methods, such as, for example, those which arecurrently employed for treating cancer and/or for preventing tumormetastasis. Preference is given to a combination with radiation therapyand chemotherapeutic agents, such as cisplatin, cyclophosphamide,5-fluorouracil, adriamycin, daunorubicin or tamoxifen. Other embodimentsinclude the use of a second dsRNA used to inhibit the expression ofVEGF.

The invention can also be practiced by including with a specific RNAiagent, in combination with another anti-cancer chemotherapeutic agent,such as any conventional chemotherapeutic agent, or another dsRNA usedto inhibit the expression of VEGF. The combination of a specific bindingagent with such other agents can potentiate the chemotherapeuticprotocol. Numerous chemotherapeutic protocols will present themselves inthe mind of the skilled practitioner as being capable of incorporationinto the method of the invention. Any chemotherapeutic agent can beused, including alkylating agents, antimetabolites, hormones andantagonists, radioisotopes, as well as natural products. For example,the compound of the invention can be administered with antibiotics suchas doxorubicin and other anthracycline analogs, nitrogen mustards suchas cyclophosphamide, pyrimidine analogs such as 5-fluorouracil,cisplatin, hydroxyurea, taxol and its natural and synthetic derivatives,and the like. As another example, in the case of mixed tumors, such asadenocarcinoma of the breast, where the tumors includegonadotropin-dependent and gonadotropin-independent cells, the compoundcan be administered in conjunction with leuprolide or goserelin(synthetic peptide analogs of LH-RH). Other antineoplastic protocolsinclude the use of a tetracycline compound with another treatmentmodality, e.g., surgery, radiation, etc., also referred to herein as“adjunct antineoplastic modalities.” Thus, the method of the inventioncan be employed with such conventional regimens with the benefit ofreducing side effects and enhancing efficacy.

Methods for Inhibiting Expression of the Eg5 Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of the Eg5 gene in a mammal. The method comprisesadministering a composition of the invention to the mammal such thatexpression of the target Eg5 gene is silenced. Because of their highspecificity, the dsRNAs of the invention specifically target RNAs(primary or processed) of the target Eg5 gene. Compositions and methodsfor inhibiting the expression of these Eg5 genes using dsRNAs can beperformed as described elsewhere herein.

In one embodiment, the method comprises administering a compositioncomprising a dsRNA, wherein the dsRNA comprises a nucleotide sequencewhich is complementary to at least a part of an RNA transcript of theEg5 gene of the mammal to be treated. When the organism to be treated isa mammal such as a human, the composition may be administered by anymeans known in the art including, but not limited to oral or parenteralroutes, including intravenous, intramuscular, subcutaneous, transdermal,airway (aerosol), nasal, rectal, and topical (including buccal andsublingual) administration. In preferred embodiments, the compositionsare administered by intravenous infusion or injection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES Gene Walking of the Eg5 Gene

Initial Screening Set

siRNA design was carried out to identify siRNAs targeting Eg5 (alsoknown as KIF11, HSKP, KNSL1 and TRIP5). Human mRNA sequences to Eg5,RefSeq ID number:NM_(—)004523, was used.

siRNA duplexes cross-reactive to human and mouse Eg5 were designed.Twenty-four duplexes were synthesized for screening. (Table 1).

Expanded Screening Set

A second screening set was defined with 266 siRNAs targeting human EG5,as well as its rhesus monkey ortholog (Table 2). An expanded screeningset was selected with 328 siRNA targeting human EG5, with no necessityto hit any EG5 mRNA of other species (Table 3).

The sequences for human and a partial rhesus EG5 mRNAs were downloadedfrom NCBI Nucleotide database and the human sequence was further on usedas reference sequence (Human EG5:NM_(—)004523.2, 4908 bp, and RhesusEG5: XM_(—)001087644.1, 878 bp (only 5′ part of human EG5)

For identification of further rhesus EG5 sequences a mega blast searchwith the human sequence was conducted at NCBI against rhesus referencegenome. The downloaded rhesus sequence and the hit regions in the blasthit were assembled to a rhesus consensus sequence with ˜92% identity tohuman EG5 over the full-length.

All possible 19mers were extracted from the human mRNA sequence,resulting in the pool of candidate target sites corresponding to 4890(sense strand) sequences of human-reactive EG5 siRNAs.

Human-rhesus cross-reactivity as prerequisite for in silico selection ofsiRNAs for an initial screening set out of this candidate pool. Todetermine rhesus-reactive siRNAs, each candidate siRNA target site wassearched for presence in the assembled rhesus sequence. Further, thepredicted specificity of the siRNA as criterion for selection of out thepool of human-rhesus cross-reactive siRNAs, manifested by targetinghuman EG5 mRNA sequences, but not other human mRNAs.

The specificity of an siRNA can be expressed via its potential to targetother genes, which are referred to as “off-target genes”.

For predicting the off-target potential of an siRNA, the followingassumptions were made:

-   -   1) off-target potential of a strand can be deduced from the        number and distribution of mismatches to an off-target    -   2) the most relevant off-target, that is the gene predicted to        have the highest probability to be silenced due to tolerance of        mismatches, determines the off-target potential of the strand    -   3) positions 2 to 9 (counting 5′ to 3′) of a strand (seed        region) may contribute more to off-target potential than rest of        sequence (that is non-seed and cleavage site region)    -   4) positions 10 and 11 (counting 5′ to 3′) of a strand (cleavage        site region) may contribute more to off-target potential than        non-seed region (that is positions 12 to 18, counting 5′ to 3′)    -   5) positions 1 and 19 of each strand are not relevant for        off-target interactions    -   6) off-target potential can be expressed by the off-target score        of the most relevant off-target, calculated based on number and        position of mismatches of the strand to the most homologous        region in the off-target gene considering assumptions 3 to 5    -   7) off-target potential of antisense and sense strand will be        relevant, whereas potential abortion of sense strand activity by        internal modifications introduced is likely

SiRNAs with low off-target potential were defined as preferable andassumed to be more specific.

In order to identify human EG5-specific siRNAs, all other humantranscripts, which were all considered potential off-targets, weresearched for potential target regions for human-rhesus cross-reactive19mer sense strand sequences as well as complementary antisense strands.For this, the fastA algorithm was used to determine the most homologueshit region in each sequence of the human RefSeq database, which weassume to represent the comprehensive human transcriptome.

To rank all potential off-targets according to assumptions 3 to 5, andby this identify the most relevant off-target gene and its off-targetscore, fastA output files were analyzed further by a perl script.

The script extracted the following off-target properties for each 19merinput sequence and each off-target gene to calculate the off-targetscore:

Number of mismatches in non-seed region

Number of mismatches in seed region

Number of mismatches in cleavage site region

The off-target score was calculated by considering assumptions 3 to 5 asfollows:

Off-target  score = number  of  seed  mismatches * 10 + number  of  cleavage  site  mismatches * 1.2 + number  of  non-seed  mismatches * 1

The most relevant off-target gene for each 19mer sequence was defined asthe gene with the lowest off-target score. Accordingly, the lowestoff-target score was defined as representative for the off-targetpotential of a strand.

For the screening set in Table 2, an off-target score of 3 or more forthe antisense strand and 2 or more for the sense strand was chosen asprerequisite for selection of siRNAs, whereas all sequences containing 4or more consecutive G's (poly-G sequences) were excluded. 266human-rhesus cross-reactive sequences passing the specificity criterion,were selected based on this cut-off (see Table 2).

For definition of the expanded screening set the cross-reactivity torhesus was disgarded, re-calculated the predicted specificity based onthe newly available human RefSeq database and selected only those 328non-poly-G siRNAs with off-target score of 2,2 or more for the antisenseand sense strand (see Table 3).

For the Tables: Key: A,G,C,U-ribonucleotides: T-deoxythymidine:u,c-2′-O-methyl nucleotides: s-phosphorothioate linkage.

TABLE 1A TABLE 1A position SEQ SEQ SEQ in human ID sequence of total IDsense sequence ID antisense sequence duplex access. # NO23mer target site No (5′-3′) No (5′-3′) name 385-407 1244ACCGAAGUGUUGUUUGUCCAAUU  1 cGAAGuGuuGuuuGuccAATsT  2UUGGAcAAAcAAcACUUCGTsT AL-DP- 6226 347-369 1245 UAUGGUGUUUGGAGCAUCUACUA 3 uGGuGuuuGGAGcAucuAcTsT  4 GuAGAUGCUCcAAAcACcATsT AL-DP- 62271078-1100 1246 AAUCUAAACUAACUAGAAUCCUC  5 ucuAAAcuAAcuAGAAuccTsT  6GGAUUCuAGUuAGUUuAGATsT AL-DP- 6228 1067-1089 1247UCCUUAUCGAGAAUCUAAACUAA  7 cuuAucGAGAAucuAAAcuTsT  8AGUUuAGAUUCUCGAuAAGTsT AL-DP- 6229 374-396 1248 GAUUGAUGUUUACCGAAGUGUUG 9 uuGAuGuuuAccGAAGuGuTsT 10 AcACUUCGGuAAAcAUcAATsT AL-DP- 6230 205-2271249 UGGUGAGAUGCAGACCAUUUAAU 11 GuGAGAuGcAGAccAuuuATsT 12uAAAUGGUCUGcAUCUcACTsT AL-DP- 6231 1176-1198 1250ACUCUGAGUACAUUGGAAUAUGC 13 ucuGAGuAcAuuGGAAuAuTsT 14AuAUUCcAAUGuACUcAGATsT AL-DP- 6232 386-408 1251 CCGAAGUGUUGUUUGUCCAAUUC15 GAAGuGuuGuuuGuccAAuTsT 16 AUUGGAcAAAcAAcACUUCTsT AL-DP- 6233 416-4381252 AGUUAUUAUGGGCUAUAAUUGCA 17 uuAuuAuGGGcuAuAAuuGTsT 18cAAUuAuAGCCcAuAAuAATsT AL-DP- 6234 485-507 1253 GGAAGGUGAAAGGUCACCUAAUG19 AAGGuGAAAGGucAccuAATsT 20 UuAGGUGACCUUUcACCUUTsT AL-DP- 6235 476-4981254 UUUUACAAUGGAAGGUGAAAGGU 21 uuAcAAuGGAAGGuGAAAGTsT 22CUUUcACCUUCcAUUGuAATsT AL-DP- 6236 486-508 1255 GAAGGUGAAAGGUCACCUAAUGA23 AGGuGAAAGGucAccuAAuTsT 24 AUuAGGUGACCUUUcACCUTsT AL-DP- 6237 487-5091256 AAGGUGAAAGGUCACCUAAUGAA 25 GGuGAAAGGucAccuAAuGTsT 26cAUuAGGUGACCUUUcACCTsT AL-DP- 6238 1066-1088 1257UUCCUUAUCGAGAAUCUAAACUA 27 ccuuAucGAGAAucuAAAcTsT 28GUUuAGAUUCUCGAuAAGGTsT AL-DP- 6239 1256-1278 1258AGCUCUUAUUAAGGAGUAUACGG 29 cucuuAuuAAGGAGuAuAcTsT 30GuAuACUCCUuAAuAAGAGTsT AL-DP- 6240 2329-2351 1259CAGAGAGAUUCUGUGCUUUGGAG 31 GAGAGAuucuGuGcuuuGGTsT 32CcAAAGcAcAGAAUCUCUCTsT AL-DP- 6241 1077-1099 1260GAAUCUAAACUAACUAGAAUCCU 33 AucuAAAcuAAcuAGAAucTsT 34GAUUCuAGUuAGUUuAGAUTsT AL-DP- 6242 1244-1266 1261ACUCACCAAAAAAGCUCUUAUUA 35 ucAccAAAAAAGcucuuAuTsT 36AuAAGAGCUUUUUUGGUGATsT AL-DP- 6243 637-659 1262 AAGAGCUUUUUGAUCUUCUUAAU37 GAGcuuuuuGAucuucuuATsT 38 uAAGAAGAUcAAAAAGCUCTsT AL-DP- 62441117-1139 1263 GGCGUACAAGAACAUCUAUAAUU 39 cGuAcAAGAAcAucuAuAATsT 40UuAuAGAUGUUCUUGuACGTsT AL-DP- 6245 373-395 1264 AGAUUGAUGUUUACCGAAGUGUU41 AuuGAuGuuuAccGAAGuGTsT 42 cACUUCGGuAAAcAUcAAUTsT AL-DP- 62461079-1101 1265 AUCUAAACUAACUAGAAUCCUCC 43 cuAAAcuAAcuAGAAuccuTsT 44AGGAUUCuAGUuAGUUuAGTsT AL-DP- 6247 383-405 1266 UUACCGAAGUGUUGUUUGUCCAA45 AccGAAGuGuuGuuuGuccTsT 46 GGAcAAAcAAcACUUCGGUTsT AL-DP- 6248 200-2221267 GGUGGUGGUGAGAUGCAGACCAU 47 uGGuGGuGAGAuGcAGAccTsT 48GGUCUGcAUCUcACcACcATsT AL-DP- 6249

TABLE 1B TABLE 1B single dose screen @ 25 nM [% SDs 2nd screen duplexresidual (among name mRNA] quadruplicates) AL-DP-6226  23%  3%AL-DP-6227  69% 10% AL-DP-6228  33%  2% AL-DP-6229   2%  2% AL-DP-6230 66% 11% AL-DP-6231  17%  1% AL-DP-6232   9%  3% AL-DP-6233  24%  6%AL-DP-6234  91%  2% AL-DP-6235 112%  4% AL-DP-6236  69%  4% AL-DP-6237 42%  2% AL-DP-6238  45%  2% AL-DP-6239   2%  1% AL-DP-6240  48%  2%AL-DP-6241  41%  2% AL-DP-6242   8%  2% AL-DP-6243   7%  1% AL-DP-6244  6%  2% AL-DP-6245  12%  2% AL-DP-6246  28%  3% AL-DP-6247  71%  4%AL-DP-6248   5%  2% AL-DP-6249  28%  3%

TABLE 2A TABLE 2A position SEQ SEQ SEQ in human ID sequence of total IDsense sequence ID antisense sequence duplex access. # NO19mer target site NO (5′-3′) NO (5′-3′) name 829-847 1268CAUACUCUAGUCGUUCCCA  49 cAuAcucuAGucGuucccATsT  50UGGGAACGACuAGAGuAUGTsT AD- 12072 246-264 1269 AGCGCCCAUUCAAUAGUAG  51AGcGcccAuucAAuAGuAGTsT  52 CuACuAUUGAAUGGGCGCUTsT AD- 12073 238-256 1270GGAAAGCUAGCGCCCAUUC  53 GGAAAGcuAGcGcccAuucTsT  54GAAUGGGCGCuAGCUUUCCTsT AD- 12074 239-257 1271 GAAAGCUAGCGCCCAUUCA  55GAAAGcuAGcGcccAuucATsT  56 UGAAUGGGCGCuAGCUUUCTsT AD- 12075 878-896 1272AGAAACUACGAUUGAUGGA  57 AGAAAcuAcGAuuGAuGGATsT  58UCcAUcAAUCGuAGUUUCUTsT AD- 12076 1064-1082 1273 UGUUCCUUAUCGAGAAUCU  59uGuuccuuAucGAGAAucuTsT  60 AGAUUCUCGAuAAGGAAcATsT AD- 12077 3278-32961274 CAGAUUACCUCUGCGAGCC  61 cAGAuuAccucuGcGAGccTsT  62GGCUCGcAGAGGuAAUCUGTsT AD- 12078 247-265 1275 GCGCCCAUUCAAUAGUAGA  63GcGcccAuucAAuAGuAGATsT  64 UCuACuAUUGAAUGGGCGCTsT AD- 12079 434-452 1276UUGCACUAUCUUUGCGUAU  65 uuGcAcuAucuuuGcGuAuTsT  66AuACGcAAAGAuAGUGcAATsT AD- 12080 232-250 1277 CAGAGCGGAAAGCUAGCGC  67cAGAGcGGAAAGcuAGcGcTsT  68 GCGCuAGCUUUCCGCUCUGTsT AD- 12081 1831-18491278 AGACCUUAUUUGGUAAUCU  69 AGAccuuAuuuGGuAAucuTsT  70AGAUuACcAAAuAAGGUCUTsT AD- 12082 1105-1123 1279 AUUCUCUUGGAGGGCGUAC  71AuucucuuGGAGGGcGuAcTsT  72 GuACGCCCUCcAAGAGAAUTsT AD- 12083 536-554 1280GGCUGGUAUAAUUCCACGU  73 GGcuGGuAuAAuuccAcGuTsT  74ACGUGGAAUuAuACcAGCCTsT AD- 12084 236-254 1281 GCGGAAAGCUAGCGCCCAU  75GcGGAAAGcuAGcGcccAuTsT  76 AUGGGCGCuAGCUUUCCGCTsT AD- 12085 435-453 1282UGCACUAUCUUUGCGUAUG  77 uGcAcuAucuuuGcGuAuGTsT  78cAuACGcAAAGAuAGUGcATsT AD- 12086 541-559 1283 GUAUAAUUCCACGUACCCU  79GuAuAAuuccAcGuAcccuTsT  80 AGGGuACGUGGAAUuAuACTsT AD- 12087 1076-10941284 AGAAUCUAAACUAACUAGA  81 AGAAucuAAAcuAAcuAGATsT  82UCuAGUuAGUUuAGAUUCUTsT AD- 12088 1432-1450 1285 AGGAGCUGAAUAGGGUUAC  83AGGAGcuGAAuAGGGuuAcTsT  84 GuAACCCuAUUcAGCUCCUTsT AD- 12089 1821-18391286 GAAGUACAUAAGACCUUAU  85 GAAGuAcAuAAGAccuuAuTsT  86AuAAGGUCUuAUGuACUUCTsT AD- 12090 2126-2144 1287 GACAGUGGCCGAUAAGAUA  87GAcAGuGGccGAuAAGAuATsT  88 uAUCUuAUCGGCcACUGUCTsT AD- 12091 2373-23911288 AAACCACUUAGUAGUGUCC  89 AAAccAcuuAGuAGuGuccTsT  90GGAcACuACuAAGUGGUUUTsT AD- 12092 4026-4044 1289 UCCCUAGACUUCCCUAUUU  91ucccuAGAcuucccuAuuuTsT  92 AAAuAGGGAAGUCuAGGGATsT AD- 12093 4030-40481290 UAGACUUCCCUAUUUCGCU  93 uAGAcuucccuAuuucGcuTsT  94AGCGAAAuAGGGAAGUCuATsT AD- 12094 144-162 1291 GCGUCGCAGCCAAAUUCGU  95GcGucGcAGccAAAuucGuTsT  96 ACGAAUUUGGCUGCGACGCTsT AD- 12095 242-260 1292AGCUAGCGCCCAUUCAAUA  97 AGcuAGcGcccAuucAAuATsT  98uAUUGAAUGGGCGCuAGCUTsT AD- 12096 879-897 1293 GAAACUACGAUUGAUGGAG  99GAAAcuAcGAuuGAuGGAGTsT 100 CUCcAUcAAUCGuAGUUUCTsT AD- 12097 2134-21521294 CCGAUAAGAUAGAAGAUCA 101 ccGAuAAGAuAGAAGAucATsT 102UGAUCUUCuAUCUuAUCGGTsT AD- 12098 245-263 1295 UAGCGCCCAUUCAAUAGUA 103uAGcGcccAuucAAuAGuATsT 104 uACuAUUGAAUGGGCGCuATsT AD- 12099 444-462 1296UUUGCGUAUGGCCAAACUG 105 uuuGcGuAuGGccAAAcuGTsT 106cAGUUUGGCcAuACGcAAATsT AD- 12100 550-568 1297 CACGUACCCUUCAUCAAAU 107cAcGuAcccuucAucAAAuTsT 108 AUUUGAUGAAGGGuACGUGTsT AD- 12101 442-460 1298UCUUUGCGUAUGGCCAAAC 109 ucuuuGcGuAuGGccAAAcTsT 110GUUUGGCcAuACGcAAAGATsT AD- 12102 386-404 1299 CCGAAGUGUUGUUUGUCCA 111ccGAAGuGuuGuuuGuccATsT 112 UGGAcAAAcAAcACUUCGGTsT AD- 12103 233-251 1300AGAGCGGAAAGCUAGCGCC 113 AGAGcGGAAAGcuAGcGccTsT 114GGCGCuAGCUUUCCGCUCUTsT AD- 12104 243-261 1301 GCUAGCGCCCAUUCAAUAG 115GcuAGcGcccAuucAAuAGTsT 116 CuAUUGAAUGGGCGCuAGCTsT AD- 12105 286-304 1302AAGUUAGUGUACGAACUGG 117 AAGuuAGuGuAcGAAcuGGTsT 118CcAGUUCGuAcACuAACUUTsT AD- 12106 294-312 1303 GUACGAACUGGAGGAUUGG 119GuAcGAAcuGGAGGAuuGGTsT 120 CcAAUCCUCcAGUUCGuACTsT AD- 12107 296-314 1304ACGAACUGGAGGAUUGGCU 121 AcGAAcuGGAGGAuuGGcuTsT 122AGCcAAUCCUCcAGUUCGUTsT AD- 12108 373-391 1305 AGAUUGAUGUUUACCGAAG 123AGAuuGAuGuuuAccGAAGTsT 124 CUUCGGuAAAcAUcAAUCUTsT AD- 12109 422-440 1306UAUGGGCUAUAAUUGCACU 125 uAuGGGcuAuAAuuGcAcuTsT 126AGUGcAAUuAuAGCCcAuATsT AD- 12110 441-459 1307 AUCUUUGCGUAUGGCCAAA 127AucuuuGcGuAuGGccAAATsT 128 UUUGGCcAuACGcAAAGAUTsT AD- 12111 832-850 1308ACUCUAGUCGUUCCCACUC 129 AcucuAGucGuucccAcucTsT 130GAGUGGGAACGACuAGAGUTsT AD- 12112 881-899 1309 AACUACGAUUGAUGGAGAA 131AAcuAcGAuuGAuGGAGAATsT 132 UUCUCcAUcAAUCGuAGUUTsT AD- 12113 975-993 1310GAUAAGAGAGCUCGGGAAG 133 GAuAAGAGAGcucGGGAAGTsT 134CUUCCCGAGCUCUCUuAUCTsT AD- 12114 1073-1091 1311 UCGAGAAUCUAAACUAACU 135ucGAGAAucuAAAcuAAcuTsT 136 AGUuAGUUuAGAUUCUCGATsT AD- 12115 1084-11021312 AACUAACUAGAAUCCUCCA 137 AAcuAAcuAGAAuccuccATsT 138UGGAGGAUUCuAGUuAGUUTsT AD- 12116 1691-1709 1313 GGAUCGUAAGAAGGCAGUU 139GGAucGuAAGAAGGcAGuuTsT 140 AACUGCCUUCUuACGAUCCTsT AD- 12117 1693-17111314 AUCGUAAGAAGGCAGUUGA 141 AucGuAAGAAGGcAGuuGATsT 142UcAACUGCCUUCUuACGAUTsT AD- 12118 1702-1720 1315 AGGCAGUUGACCAACACAA 143AGGcAGuuGAccAAcAcAATsT 144 UUGUGUUGGUcAACUGCCUTsT AD- 12119 2131-21491316 UGGCCGAUAAGAUAGAAGA 145 uGGccGAuAAGAuAGAAGATsT 146UCUUCuAUCUuAUCGGCcATsT AD- 12120 2412-2430 1317 UCUAAGGAUAUAGUCAACA 147ucuAAGGAuAuAGucAAcATsT 148 UGUUGACuAuAUCCUuAGATsT AD- 12121 2859-28771318 ACUAAGCUUAAUUGCUUUC 149 AcuAAGcuuAAuuGcuuucTsT 150GAAAGcAAUuAAGCUuAGUTsT AD- 12122 3294-3312 1319 GCCCAGAUCAACCUUUAAU 151GcccAGAucAAccuuuAAuTsT 152 AUuAAAGGUUGAUCUGGGCTsT AD- 12123 223-241 1320UUAAUUUGGCAGAGCGGAA 153 uuAAuuuGGcAGAGcGGAATsT 154UUCCGCUCUGCcAAAUuAATsT AD- 12124 1070-1088 1321 UUAUCGAGAAUCUAAACUA 155uuAucGAGAAucuAAAcuATsT 156 uAGUUuAGAUUCUCGAuAATsT AD- 12125 244-262 1322CUAGCGCCCAUUCAAUAGU 157 cuAGcGcccAuucAAuAGuTsT 158ACuAUUGAAUGGGCGCuAGTsT AD- 12126 257-275 1323 AAUAGUAGAAUGUGAUCCU 159AAuAGuAGAAuGuGAuccuTsT 160 AGGAUcAcAUUCuACuAUUTsT AD- 12127 277-295 1324UACGAAAAGAAGUUAGUGU 161 uAcGAAAAGAAGuuAGuGuTsT 162AcACuAACUUCUUUUCGuATsT AD- 12128 284-302 1325 AGAAGUUAGUGUACGAACU 163AGAAGuuAGuGuAcGAAcuTsT 164 AGUUCGuAcACuAACUUCUTsT AD- 12129 366-384 1326ACUAAACAGAUUGAUGUUU 165 AcuAAAcAGAuuGAuGuuuTsT 166AAAcAUcAAUCUGUUuAGUTsT AD- 12130 443-461 1327 CUUUGCGUAUGGCCAAACU 167cuuuGcGuAuGGccAAAcuTsT 168 AGUUUGGCcAuACGcAAAGTsT AD- 12131 504-522 1328AAUGAAGAGUAUACCUGGG 169 AAuGAAGAGuAuAccuGGGTsT 170CCcAGGuAuACUCUUcAUUTsT AD- 12132 543-561 1329 AUAAUUCCACGUACCCUUC 171AuAAuuccAcGuAcccuucTsT 172 GAAGGGuACGUGGAAUuAUTsT AD- 12133 551-569 1330ACGUACCCUUCAUCAAAUU 173 AcGuAcccuucAucAAAuuTsT 174AAUUUGAUGAAGGGuACGUTsT AD- 12134 552-570 1331 CGUACCCUUCAUCAAAUUU 175cGuAcccuucAucAAAuuuTsT 176 AAAUUUGAUGAAGGGuACGTsT AD- 12135 553-571 1332GUACCCUUCAUCAAAUUUU 177 GuAcccuucAucAAAuuuuTsT 178AAAAUUUGAUGAAGGGuACTsT AD- 12136 577-595 1333 AACUUACUGAUAAUGGUAC 179AAcuuAcuGAuAAuGGuAcTsT 180 GuACcAUuAUcAGuAAGUUTsT AD- 12137 602-620 1334UUCAGUCAAAGUGUCUCUG 181 uucAGucAAAGuGucucuGTsT 182cAGAGAcACUUUGACUGAATsT AD- 12138 652-670 1335 UUCUUAAUCCAUCAUCUGA 183uucuuAAuccAucAucuGATsT 184 UcAGAUGAUGGAUuAAGAATsT AD- 12139 747-765 1336ACAGUACACAACAAGGAUG 185 AcAGuAcAcAAcAAGGAuGTsT 186cAUCCUUGUUGUGuACUGUTsT AD- 12140 877-895 1337 AAGAAACUACGAUUGAUGG 187AAGAAAcuAcGAuuGAuGGTsT 188 CcAUcAAUCGuAGUUUCUUTsT AD- 12141 880-898 1338AAACUACGAUUGAUGGAGA 189 AAAcuAcGAuuGAuGGAGATsT 190UCUCcAUcAAUCGuAGUUUTsT AD- 12142 965-983 1339 UGGAGCUGUUGAUAAGAGA 191uGGAGcuGuuGAuAAGAGATsT 192 UCUCUuAUcAAcAGCUCcATsT AD- 12143 1086-11041340 CUAACUAGAAUCCUCCAGG 193 cuAAcuAGAAuccuccAGGTsT 194CCUGGAGGAUUCuAGUuAGTsT AD- 12144 1191-1209 1341 GAAUAUGCUCAUAGAGCAA 195GAAuAuGcucAuAGAGcAATsT 196 UUGCUCuAUGAGcAuAUUCTsT AD- 12145 1195-12131342 AUGCUCAUAGAGCAAAGAA 197 AuGcucAuAGAGcAAAGAATsT 198UUCUUUGCUCuAUGAGcAUTsT AD- 12146 1412-1430 1343 AAAAAUUGGUGCUGUUGAG 199AAAAAuuGGuGcuGuuGAGTsT 200 CUcAAcAGcACcAAUUUUUTsT AD- 12147 1431-14491344 GAGGAGCUGAAUAGGGUUA 201 GAGGAGcuGAAuAGGGuuATsT 202uAACCCuAUUcAGCUCCUCTsT AD- 12148 1433-1451 1345 GGAGCUGAAUAGGGUUACA 203GGAGcuGAAuAGGGuuAcATsT 204 UGuAACCCuAUUcAGCUCCTsT AD- 12149 1434-14521346 GAGCUGAAUAGGGUUACAG 205 GAGcuGAAuAGGGuuAcAGTsT 206CUGuAACCCuAUUcAGCUCTsT AD- 12150 1435-1453 1347 AGCUGAAUAGGGUUACAGA 207AGcuGAAuAGGGuuAcAGATsT 208 UCUGuAACCCuAUUcAGCUTsT AD- 12151 1436-14541348 GCUGAAUAGGGUUACAGAG 209 GcuGAAuAGGGuuAcAGAGTsT 210CUCUGuAACCCuAUUcAGCTsT AD- 12152 1684-1702 1349 CCAAACUGGAUCGUAAGAA 211ccAAAcuGGAucGuAAGAATsT 212 UUCUuACGAUCcAGUUUGGTsT AD- 12153 1692-17101350 GAUCGUAAGAAGGCAGUUG 213 GAucGuAAGAAGGcAGuuGTsT 214cAACUGCCUUCUuACGAUCTsT AD- 12154 1833-1851 1351 ACCUUAUUUGGUAAUCUGC 215AccuuAuuuGGuAAucuGcTsT 216 GcAGAUuACcAAAuAAGGUTsT AD- 12155 1872-18901352 UUAGAUACCAUUACUACAG 217 uuAGAuAccAuuAcuAcAGTsT 218CUGuAGuAAUGGuAUCuAATsT AD- 12156 1876-1894 1353 AUACCAUUACUACAGUAGC 219AuAccAuuAcuAcAGuAGcTsT 220 GCuACUGuAGuAAUGGuAUTsT AD- 12157 1883-19011354 UACUACAGUAGCACUUGGA 221 uAcuAcAGuAGcAcuuGGATsT 222UCcAAGUGCuACUGuAGuATsT AD- 12158 1987-2005 1355 AAAGUAAAACUGUACUACA 223AAAGuAAAAcuGuAcuAcATsT 224 UGuAGuAcAGUUUuACUUUTsT AD- 12159 2022-20401356 CUCAAGACUGAUCUUCUAA 225 cucAAGAcuGAucuucuAATsT 226UuAGAAGAUcAGUCUUGAGTsT AD- 12160 2124-2142 1357 UUGACAGUGGCCGAUAAGA 227uuGAcAGuGGccGAuAAGATsT 228 UCUuAUCGGCcACUGUcAATsT AD- 12161 2125-21431358 UGACAGUGGCCGAUAAGAU 229 uGAcAGuGGccGAuAAGAuTsT 230AUCUuAUCGGCcACUGUcATsT AD- 12162 2246-2264 1359 GCAAUGUGGAAACCUAACU 231GcAAuGuGGAAAccuAAcuTsT 232 AGUuAGGUUUCcAcAUUGCTsT AD- 12163 2376-23941360 CCACUUAGUAGUGUCCAGG 233 ccAcuuAGuAGuGuccAGGTsT 234CCUGGAcACuACuAAGUGGTsT AD- 12164 2504-2522 1361 AGAAGGUACAAAAUUGGUU 235AGAAGGuAcAAAAuuGGuuTsT 236 AACcAAUUUUGuACCUUCUTsT AD- 12165 2852-28701362 UGGUUUGACUAAGCUUAAU 237 uGGuuuGAcuAAGcuuAAuTsT 238AUuAAGCUuAGUcAAACcATsT AD- 12166 2853-2871 1363 GGUUUGACUAAGCUUAAUU 239GGuuuGAcuAAGcuuAAuuTsT 240 AAUuAAGCUuAGUcAAACCTsT AD- 12167 3110-31281364 UCUAAGUCAAGAGCCAUCU 241 ucuAAGucAAGAGccAucuTsT 242AGAUGGCUCUUGACUuAGATsT AD- 12168 3764-3782 1365 UCAUCCCUAUAGUUCACUU 243ucAucccuAuAGuucAcuuTsT 244 AAGUGAACuAuAGGGAUGATsT AD- 12169 3765-37831366 CAUCCCUAUAGUUCACUUU 245 cAucccuAuAGuucAcuuuTsT 246AAAGUGAACuAuAGGGAUGTsT AD- 12170 4027-4045 1367 CCCUAGACUUCCCUAUUUC 247cccuAGAcuucccuAuuucTsT 248 GAAAuAGGGAAGUCuAGGGTsT AD- 12171 4031-40491368 AGACUUCCCUAUUUCGCUU 249 AGAcuucccuAuuucGcuuTsT 250AAGCGAAAuAGGGAAGUCUTsT AD- 12172 4082-4100 1369 UCACCAAACCAUUUGUAGA 251ucAccAAAccAuuuGuAGATsT 252 UCuAcAAAUGGUUUGGUGATsT AD- 12173 4272-42901370 UCCUUUAAGAGGCCUAACU 253 uccuuuAAGAGGccuAAcuTsT 254AGUuAGGCCUCUuAAAGGATsT AD- 12174 4275-4293 1371 UUUAAGAGGCCUAACUCAU 255uuuAAGAGGccuAAcucAuTsT 256 AUGAGUuAGGCCUCUuAAATsT AD- 12175 4276-42941372 UUAAGAGGCCUAACUCAUU 257 uuAAGAGGccuAAcucAuuTsT 258AAUGAGUuAGGCCUCUuAATsT AD- 12176 4282-4300 1373 GGCCUAACUCAUUCACCCU 259GGccuAAcucAuucAcccuTsT 260 AGGGUGAAUGAGUuAGGCCTsT AD- 12177 4571-45891374 UGGUAUUUUUGAUCUGGCA 261 uGGuAuuuuuGAucuGGcATsT 262UGCcAGAUcAAAAAuACcATsT AD- 12178 4677-4695 1375 AGUUUAGUGUGUAAAGUUU 263AGuuuAGuGuGuAAAGuuuTsT 264 AAACUUuAcAcACuAAACUTsT AD- 12179 152-170 1376GCCAAAUUCGUCUGCGAAG 265 GccAAAuucGucuGcGAAGTsT 266CUUCGcAGACGAAUUUGGCTsT AD- 12180 156-174 1377 AAUUCGUCUGCGAAGAAGA 267AAuucGucuGcGAAGAAGATsT 268 UCUUCUUCGcAGACGAAUUTsT AD- 12181 491-509 1378UGAAAGGUCACCUAAUGAA 269 uGAAAGGucAccuAAuGAATsT 270UUcAUuAGGUGACCUUUcATsT AD- 12182 215-233 1379 CAGACCAUUUAAUUUGGCA 271cAGAccAuuuAAuuuGGcATsT 272 UGCcAAAUuAAAUGGUCUGTsT AD- 12183 216-234 1380AGACCAUUUAAUUUGGCAG 273 AGAccAuuuAAuuuGGcAGTsT 274CUGCcAAAUuAAAUGGUCUTsT AD- 12184 416-434 1381 AGUUAUUAUGGGCUAUAAU 275AGuuAuuAuGGGcuAuAAuTsT 276 AUuAuAGCCcAuAAuAACUTsT AD- 12185 537-555 1382GCUGGUAUAAUUCCACGUA 277 GcuGGuAuAAuuccAcGuATsT 278uACGUGGAAUuAuACcAGCTsT AD- 12186 221-239 1383 AUUUAAUUUGGCAGAGCGG 279AuuuAAuuuGGcAGAGcGGTsT 280 CCGCUCUGCcAAAUuAAAUTsT AD- 12187 222-240 1384UUUAAUUUGGCAGAGCGGA 281 uuuAAuuuGGcAGAGcGGATsT 282UCCGCUCUGCcAAAUuAAATsT AD- 12188 227-245 1385 UUUGGCAGAGCGGAAAGCU 283uuuGGcAGAGcGGAAAGcuTsT 284 AGCUUUCCGCUCUGCcAAATsT AD- 12189 476-494 1386UUUUACAAUGGAAGGUGAA 285 uuuuAcAAuGGAAGGuGAATsT 286UUcACCUUCcAUUGuAAAATsT AD- 12190 482-500 1387 AAUGGAAGGUGAAAGGUCA 287AAuGGAAGGuGAAAGGucATsT 288 UGACCUUUcACCUUCcAUUTsT AD- 12191 208-226 1388UGAGAUGCAGACCAUUUAA 289 uGAGAuGcAGAccAuuuAATsT 290UuAAAUGGUCUGcAUCUcATsT AD- 12192 147-165 1389 UCGCAGCCAAAUUCGUCUG 291ucGcAGccAAAuucGucuGTsT 292 cAGACGAAUUUGGCUGCGATsT AD- 12193 426-444 1390GGCUAUAAUUGCACUAUCU 293 GGcuAuAAuuGcAcuAucuTsT 294AGAuAGUGcAAUuAuAGCCTsT AD- 12194 2123-2141 1391 AUUGACAGUGGCCGAUAAG 295AuuGAcAGuGGccGAuAAGTsT 296 CUuAUCGGCcACUGUcAAUTsT AD- 12195 4029-40471392 CUAGACUUCCCUAUUUCGC 297 cuAGAcuucccuAuuucGcTsT 298GCGAAAuAGGGAAGUCuAGTsT AD- 12196 438-456 1393 ACUAUCUUUGCGUAUGGCC 299AcuAucuuuGcGuAuGGccTsT 300 GGCcAuACGcAAAGAuAGUTsT AD- 12197 830-848 1394AUACUCUAGUCGUUCCCAC 301 AuAcucuAGucGuucccAcTsT 302GUGGGAACGACuAGAGuAUTsT AD- 12198 876-894 1395 AAAGAAACUACGAUUGAUG 303AAAGAAAcuAcGAuuGAuGTsT 304 cAUcAAUCGuAGUUUCUUUTsT AD- 12199 115-133 1396GCCUUGAUUUUUUGGCGGG 305 GccuuGAuuuuuuGGcGGGTsT 306CCCGCcAAAAAAUcAAGGCTsT AD- 12200 248-266 1397 CGCCCAUUCAAUAGUAGAA 307cGcccAuucAAuAGuAGAATsT 308 UUCuACuAUUGAAUGGGCGTsT AD- 12201 1834-18521398 CCUUAUUUGGUAAUCUGCU 309 ccuuAuuuGGuAAucuGcuTsT 310AGcAGAUuACcAAAuAAGGTsT AD- 12202 3050-3068 1399 AGAGACAAUUCCGGAUGUG 311AGAGAcAAuuccGGAuGuGTsT 312 cAcAUCCGGAAUUGUCUCUTsT AD- 12203 4705-47231400 UGACUUUGAUAGCUAAAUU 313 uGAcuuuGAuAGcuAAAuuTsT 314AAUUuAGCuAUcAAAGUcATsT AD- 12204 229-247 1401 UGGCAGAGCGGAAAGCUAG 315uGGcAGAGcGGAAAGcuAGTsT 316 CuAGCUUUCCGCUCUGCcATsT AD- 12205 234-252 1402GAGCGGAAAGCUAGCGCCC 317 GAGcGGAAAGcuAGcGcccTsT 318GGGCGCuAGCUUUCCGCUCTsT AD- 12206 282-300 1403 AAAGAAGUUAGUGUACGAA 319AAAGAAGuuAGuGuAcGAATsT 320 UUCGuAcACuAACUUCUUUTsT AD- 12207 433-451 1404AUUGCACUAUCUUUGCGUA 321 AuuGcAcuAucuuuGcGuATsT 322uACGcAAAGAuAGUGcAAUTsT AD- 12208 540-558 1405 GGUAUAAUUCCACGUACCC 323GGuAuAAuuccAcGuAcccTsT 324 GGGuACGUGGAAUuAuACCTsT AD- 12209 831-849 1406UACUCUAGUCGUUCCCACU 325 uAcucuAGucGuucccAcuTsT 326AGUGGGAACGACuAGAGuATsT AD- 12210 872-890 1407 UAUGAAAGAAACUACGAUU 327uAuGAAAGAAAcuAcGAuuTsT 328 AAUCGuAGUUUCUUUcAuATsT AD- 12211 1815-18331408 AUGCUAGAAGUACAUAAGA 329 AuGcuAGAAGuAcAuAAGATsT 330UCUuAUGuACUUCuAGcAUTsT AD- 12212 1822-1840 1409 AAGUACAUAAGACCUUAUU 331AAGuAcAuAAGAccuuAuuTsT 332 AAuAAGGUCUuAUGuACUUTsT AD- 12213 3002-30201410 ACAGCCUGAGCUGUUAAUG 333 AcAGccuGAGcuGuuAAuGTsT 334cAUuAAcAGCUcAGGCUGUTsT AD- 12214 3045-3063 1411 AAAGAAGAGACAAUUCCGG 335AAAGAAGAGAcAAuuccGGTsT 336 CCGGAAUUGUCUCUUCUUUTsT AD- 12215 3224-32421412 CACACUGGAGAGGUCUAAA 337 cAcAcuGGAGAGGucuAAATsT 338UUuAGACCUCUCcAGUGUGTsT AD- 12216 3226-3244 1413 CACUGGAGAGGUCUAAAGU 339cAcuGGAGAGGucuAAAGuTsT 340 ACUUuAGACCUCUCcAGUGTsT AD- 12217 3227-32451414 ACUGGAGAGGUCUAAAGUG 341 AcuGGAGAGGucuAAAGuGTsT 342cACUUuAGACCUCUCcAGUTsT AD- 12218 145-163 1415 CGUCGCAGCCAAAUUCGUC 343cGucGcAGccAAAuucGucTsT 344 GACGAAUUUGGCUGCGACGTsT AD- 12219 1700-17181416 GAAGGCAGUUGACCAACAC 345 GAAGGcAGuuGAccAAcAcTsT 346GUGUUGGUcAACUGCCUUCTsT AD- 12220 4291-4309 1417 CAUUCACCCUGACAGAGUU 347cAuucAcccuGAcAGAGuuTsT 348 AACUCUGUcAGGGUGAAUGTsT AD- 12221 4278-42961418 AAGAGGCCUAACUCAUUCA 349 AAGAGGccuAAcucAuucATsT 350UGAAUGAGUuAGGCCUCUUTsT AD- 12222 3051-3069 1419 GAGACAAUUCCGGAUGUGG 351GAGAcAAuuccGGAuGuGGTsT 352 CcAcAUCCGGAAUUGUCUCTsT AD- 12223 3058-30761420 UUCCGGAUGUGGAUGUAGA 353 uuccGGAuGuGGAuGuAGATsT 354UCuAcAUCcAcAUCCGGAATsT AD- 12224 241-259 1421 AAGCUAGCGCCCAUUCAAU 355AAGcuAGcGcccAuucAAuTsT 356 AUUGAAUGGGCGCuAGCUUTsT AD- 12225 285-303 1422GAAGUUAGUGUACGAACUG 357 GAAGuuAGuGuAcGAAcuGTsT 358cAGUUCGuAcACuAACUUCTsT AD- 12226 542-560 1423 UAUAAUUCCACGUACCCUU 359uAuAAuuccAcGuAcccuuTsT 360 AAGGGuACGUGGAAUuAuATsT AD- 12227 2127-21451424 ACAGUGGCCGAUAAGAUAG 361 AcAGuGGccGAuAAGAuAGTsT 362CuAUCUuAUCGGCcACUGUTsT AD- 12228 3760-3778 1425 UCUGUCAUCCCUAUAGUUC 363ucuGucAucccuAuAGuucTsT 364 GAACuAuAGGGAUGAcAGATsT AD- 12229 3993-40111426 UUCUUGCUAUGACUUGUGU 365 uucuuGcuAuGAcuuGuGuTsT 366AcAcAAGUcAuAGcAAGAATsT AD- 12230 1696-1714 1427 GUAAGAAGGCAGUUGACCA 367GuAAGAAGGcAGuuGAccATsT 368 UGGUcAACUGCCUUCUuACTsT AD- 12231 2122-21401428 CAUUGACAGUGGCCGAUAA 369 cAuuGAcAGuGGccGAuAATsT 370UuAUCGGCcACUGUcAAUGTsT AD- 12232 2371-2389 1429 AGAAACCACUUAGUAGUGU 371AGAAAccAcuuAGuAGuGuTsT 372 AcACuACuAAGUGGUUUCUTsT AD- 12233 3143-31611430 GGAUUGUUCAUCAAUUGGC 373 GGAuuGuucAucAAuuGGcTsT 374GCcAAUUGAUGAAcAAUCCTsT AD- 12234 4277-4295 1431 UAAGAGGCCUAACUCAUUC 375uAAGAGGccuAAcucAuucTsT 376 GAAUGAGUuAGGCCUCUuATsT AD- 12235 287-305 1432AGUUAGUGUACGAACUGGA 377 AGuuAGuGuAcGAAcuGGATsT 378UCcAGUUCGuAcACuAACUTsT AD- 12236 1823-1841 1433 AGUACAUAAGACCUUAUUU 379AGuAcAuAAGAccuuAuuuTsT 380 AAAuAAGGUCUuAUGuACUTsT AD- 12237 3379-33971434 UGAGCCUUGUGUAUAGAUU 381 uGAGccuuGuGuAuAGAuuTsT 382AAUCuAuAcAcAAGGCUcATsT AD- 12238 4273-4291 1435 CCUUUAAGAGGCCUAACUC 383ccuuuAAGAGGccuAAcucTsT 384 GAGUuAGGCCUCUuAAAGGTsT AD- 12239 2375-23931436 ACCACUUAGUAGUGUCCAG 385 AccAcuuAGuAGuGuccAGTsT 386CUGGAcACuACuAAGUGGUTsT AD- 12240 4439-4457 1437 GAAACUUCCAAUUAUGUCU 387GAAAcuuccAAuuAuGucuTsT 388 AGAcAuAAUUGGAAGUUUCTsT AD- 12241 827-845 1438UGCAUACUCUAGUCGUUCC 389 uGcAuAcucuAGucGuuccTsT 390GGAACGACuAGAGuAUGcATsT AD- 12242 1699-1717 1439 AGAAGGCAGUUGACCAACA 391AGAAGGcAGuuGAccAAcATsT 392 UGUUGGUcAACUGCCUUCUTsT AD- 12243 1824-18421440 GUACAUAAGACCUUAUUUG 393 GuAcAuAAGAccuuAuuuGTsT 394cAAAuAAGGUCUuAUGuACTsT AD- 12244 429-447 1441 UAUAAUUGCACUAUCUUUG 395uAuAAuuGcAcuAucuuuGTsT 396 cAAAGAuAGUGcAAUuAuATsT AD- 12245 856-874 1442UCUCUGUUACAAUACAUAU 397 ucucuGuuAcAAuAcAuAuTsT 398AuAUGuAUUGuAAcAGAGATsT AD- 12246 1194-1212 1443 UAUGCUCAUAGAGCAAAGA 399uAuGcucAuAGAGcAAAGATsT 400 UCUUUGCUCuAUGAGcAuATsT AD- 12247 392-410 1444UGUUGUUUGUCCAAUUCUG 401 uGuuGuuuGuccAAuucuGTsT 402cAGAAUUGGAcAAAcAAcATsT AD- 12248 1085-1103 1445 ACUAACUAGAAUCCUCCAG 403AcuAAcuAGAAuccuccAGTsT 404 CUGGAGGAUUCuAGUuAGUTsT AD- 12249 2069-20871446 UGUGGUGUCUAUACUGAAA 405 uGuGGuGucuAuAcuGAAATsT 406UUUcAGuAuAGAcACcAcATsT AD- 12250 4341-4359 1447 UAUUAUGGGAGACCACCCA 407uAuuAuGGGAGAccAcccATsT 408 UGGGUGGUCUCCcAuAAuATsT AD- 12251 759-777 1448AAGGAUGAAGUCUAUCAAA 409 AAGGAuGAAGucuAucAAATsT 410UUUGAuAGACUUcAUCCUUTsT AD- 12252 973-991 1449 UUGAUAAGAGAGCUCGGGA 411uuGAuAAGAGAGcucGGGATsT 412 UCCCGAGCUCUCUuAUcAATsT AD- 12253 1063-10811450 AUGUUCCUUAUCGAGAAUC 413 AuGuuccuuAucGAGAAucTsT 414GAUUCUCGAuAAGGAAcAUTsT AD- 12254 1190-1208 1451 GGAAUAUGCUCAUAGAGCA 415GGAAuAuGcucAuAGAGcATsT 416 UGCUCuAUGAGcAuAUUCCTsT AD- 12255 1679-16971452 CCAUUCCAAACUGGAUCGU 417 ccAuuccAAAcuGGAucGuTsT 418ACGAUCcAGUUUGGAAUGGTsT AD- 12256 1703-1721 1453 GGCAGUUGACCAACACAAU 419GGcAGuuGAccAAcAcAAuTsT 420 AUUGUGUUGGUcAACUGCCTsT AD- 12257 1814-18321454 CAUGCUAGAAGUACAUAAG 421 cAuGcuAGAAGuAcAuAAGTsT 422CUuAUGuACUUCuAGcAUGTsT AD- 12258 1818-1836 1455 CUAGAAGUACAUAAGACCU 423cuAGAAGuAcAuAAGAccuTsT 424 AGGUCUuAUGuACUUCuAGTsT AD- 12259 1897-19151456 UUGGAUCUCUCACAUCUAU 425 uuGGAucucucAcAucuAuTsT 426AuAGAUGUGAGAGAUCcAATsT AD- 12260 2066-2084 1457 AACUGUGGUGUCUAUACUG 427AAcuGuGGuGucuAuAcuGTsT 428 cAGuAuAGAcACcAcAGUUTsT AD- 12261 2121-21391458 UCAUUGACAGUGGCCGAUA 429 ucAuuGAcAGuGGccGAuATsT 430uAUCGGCcACUGUcAAUGATsT AD- 12262 2280-2298 1459 AUAAAGCAGACCCAUUCCC 431AuAAAGcAGAcccAuucccTsT 432 GGGAAUGGGUCUGCUUuAUTsT AD- 12263 2369-23871460 ACAGAAACCACUUAGUAGU 433 AcAGAAAccAcuuAGuAGuTsT 434ACuACuAAGUGGUUUCUGUTsT AD- 12264 2372-2390 1461 GAAACCACUUAGUAGUGUC 435GAAAccAcuuAGuAGuGucTsT 436 GAcACuACuAAGUGGUUUCTsT AD- 12265 2409-24271462 AAAUCUAAGGAUAUAGUCA 437 AAAucuAAGGAuAuAGucATsT 438UGACuAuAUCCUuAGAUUUTsT AD- 12266 2933-2951 1463 UUAUUUAUACCCAUCAACA 439uuAuuuAuAcccAucAAcATsT 440 UGUUGAUGGGuAuAAAuAATsT AD- 12267 3211-32291464 ACAGAGGCAUUAACACACU 441 AcAGAGGcAuuAAcAcAcuTsT 442AGUGUGUuAAUGCCUCUGUTsT AD- 12268 3223-3241 1465 ACACACUGGAGAGGUCUAA 443AcAcAcuGGAGAGGucuAATsT 444 UuAGACCUCUCcAGUGUGUTsT AD- 12269 3225-32431466 ACACUGGAGAGGUCUAAAG 445 AcAcuGGAGAGGucuAAAGTsT 446CUUuAGACCUCUCcAGUGUTsT AD- 12270 3291-3309 1467 CGAGCCCAGAUCAACCUUU 447cGAGcccAGAucAAccuuuTsT 448 AAAGGUUGAUCUGGGCUCGTsT AD- 12271 4036-40541468 UCCCUAUUUCGCUUUCUCC 449 ucccuAuuucGcuuucuccTsT 450GGAGAAAGCGAAAuAGGGATsT AD- 12272 4180-4198 1469 UCUAAAAUCACUGUCAACA 451ucuAAAAucAcuGucAAcATsT 452 UGUUGAcAGUGAUUUuAGATsT AD- 12273 151-169 1470AGCCAAAUUCGUCUGCGAA 453 AGccAAAuucGucuGcGAATsT 454UUCGcAGACGAAUUUGGCUTsT AD- 12274 250-268 1471 CCCAUUCAAUAGUAGAAUG 455cccAuucAAuAGuAGAAuGTsT 456 cAUUCuACuAUUGAAUGGGTsT AD- 12275 821-839 1472GAUGAAUGCAUACUCUAGU 457 GAuGAAuGcAuAcucuAGuTsT 458ACuAGAGuAUGcAUUcAUCTsT AD- 12276 1060-1078 1473 CUCAUGUUCCUUAUCGAGA 459cucAuGuuccuuAucGAGATsT 460 UCUCGAuAAGGAAcAUGAGTsT AD- 12277 1075-10931474 GAGAAUCUAAACUAACUAG 461 GAGAAucuAAAcuAAcuAGTsT 462CuAGUuAGUUuAGAUUCUCTsT AD- 12278 1819-1837 1475 UAGAAGUACAUAAGACCUU 463uAGAAGuAcAuAAGAccuuTsT 464 AAGGUCUuAUGuACUUCuATsT AD- 12279 3003-30211476 CAGCCUGAGCUGUUAAUGA 465 cAGccuGAGcuGuuAAuGATsT 466UcAUuAAcAGCUcAGGCUGTsT AD- 12280 3046-3064 1477 AAGAAGAGACAAUUCCGGA 467AAGAAGAGAcAAuuccGGATsT 468 UCCGGAAUUGUCUCUUCUUTsT AD- 12281 3134-31521478 UGCUGGUGUGGAUUGUUCA 469 uGcuGGuGuGGAuuGuucATsT 470UGAAcAAUCcAcACcAGcATsT AD- 12282 155-173 1479 AAAUUCGUCUGCGAAGAAG 471AAAuucGucuGcGAAGAAGTsT 472 CUUCUUCGcAGACGAAUUUTsT AD- 12283 4596-46141480 UUUCUGGAAGUUGAGAUGU 473 uuucuGGAAGuuGAGAuGuTsT 474AcAUCUcAACUUCcAGAAATsT AD- 12284 365-383 1481 UACUAAACAGAUUGAUGUU 475uAcuAAAcAGAuuGAuGuuTsT 476 AAcAUcAAUCUGUUuAGuATsT AD- 12285 374-392 1482GAUUGAUGUUUACCGAAGU 477 GAuuGAuGuuuAccGAAGuTsT 478ACUUCGGuAAAcAUcAAUCTsT AD- 12286 436-454 1483 GCACUAUCUUUGCGUAUGG 479GcAcuAucuuuGcGuAuGGTsT 480 CcAuACGcAAAGAuAGUGCTsT AD- 12287 539-557 1484UGGUAUAAUUCCACGUACC 481 uGGuAuAAuuccAcGuAccTsT 482GGuACGUGGAAUuAuACcATsT AD- 12288 1629-1647 1485 AGCAAGCUGCUUAACACAG 483AGcAAGcuGcuuAAcAcAGTsT 484 CUGUGUuAAGcAGCUUGCUTsT AD- 12289 2370-23881486 CAGAAACCACUUAGUAGUG 485 cAGAAAccAcuuAGuAGuGTsT 486cACuACuAAGUGGUUUCUGTsT AD- 12290 2676-2694 1487 AACUUAUUGGAGGUUGUAA 487AAcuuAuuGGAGGuuGuAATsT 488 UuAcAACCUCcAAuAAGUUTsT AD- 12291 3228-32461488 CUGGAGAGGUCUAAAGUGG 489 cuGGAGAGGucuAAAGuGGTsT 490CcACUUuAGACCUCUCcAGTsT AD- 12292 3703-3721 1489 AAAAAAGAUAUAAGGCAGU 491AAAAAAGAuAuAAGGcAGuTsT 492 ACUGCCUuAuAUCUUUUUUTsT AD- 12293 3737-37551490 GAAUUUUGAUAUCUACCCA 493 GAAuuuuGAuAucuAcccATsT 494UGGGuAGAuAUcAAAAUUCTsT AD- 12294 4573-4591 1491 GUAUUUUUGAUCUGGCAAC 495GuAuuuuuGAucuGGcAAcTsT 496 GUUGCcAGAUcAAAAAuACTsT AD- 12295 526-544 1492AGGAUCCCUUGGCUGGUAU 497 AGGAucccuuGGcuGGuAuTsT 498AuACcAGCcAAGGGAUCCUTsT AD- 12296 527-545 1493 GGAUCCCUUGGCUGGUAUA 499GGAucccuuGGcuGGuAuATsT 500 uAuACcAGCcAAGGGAUCCTsT AD- 12297 256-274 1494CAAUAGUAGAAUGUGAUCC 501 cAAuAGuAGAAuGuGAuccTsT 502GGAUcAcAUUCuACuAUUGTsT AD- 12298 427-445 1495 GCUAUAAUUGCACUAUCUU 503GcuAuAAuuGcAcuAucuuTsT 504 AAGAuAGUGcAAUuAuAGCTsT AD- 12299 554-572 1496UACCCUUCAUCAAAUUUUU 505 uAcccuucAucAAAuuuuuTsT 506AAAAAUUUGAUGAAGGGuATsT AD- 12300 1210-1228 1497 AGAACAUAUUGAAUAAGCC 507AGAAcAuAuuGAAuAAGccTsT 508 GGCUuAUUcAAuAUGUUCUTsT AD- 12301 1414-14321498 AAAUUGGUGCUGUUGAGGA 509 AAAuuGGuGcuGuuGAGGATsT 510UCCUcAAcAGcACcAAUUUTsT AD- 12302 1438-1456 1499 UGAAUAGGGUUACAGAGUU 511uGAAuAGGGuuAcAGAGuuTsT 512 AACUCUGuAACCCuAUUcATsT AD- 12303 1516-15341500 AAGAACUUGAAACCACUCA 513 AAGAAcuuGAAAccAcucATsT 514UGAGUGGUUUcAAGUUCUUTsT AD- 12304 2279-2297 1501 AAUAAAGCAGACCCAUUCC 515AAuAAAGcAGAcccAuuccTsT 516 GGAAUGGGUCUGCUUuAUUTsT AD- 12305 2939-29571502 AUACCCAUCAACACUGGUA 517 AuAcccAucAAcAcuGGuATsT 518uACcAGUGUUGAUGGGuAUTsT AD- 12306 3142-3160 1503 UGGAUUGUUCAUCAAUUGG 519uGGAuuGuucAucAAuuGGTsT 520 CcAAUUGAUGAAcAAUCcATsT AD- 12307 3229-32471504 UGGAGAGGUCUAAAGUGGA 521 uGGAGAGGucuAAAGuGGATsT 522UCcACUUuAGACCUCUCcATsT AD- 12308 3763-3781 1505 GUCAUCCCUAUAGUUCACU 523GucAucccuAuAGuucAcuTsT 524 AGUGAACuAuAGGGAUGACTsT AD- 12309 4801-48191506 AUAAUGGCUAUAAUUUCUC 525 AuAAuGGcuAuAAuuucucTsT 526GAGAAAUuAuAGCcAUuAUTsT AD- 12310 529-547 1507 AUCCCUUGGCUGGUAUAAU 527AucccuuGGcuGGuAuAAuTsT 528 AUuAuACcAGCcAAGGGAUTsT AD- 12311 425-443 1508GGGCUAUAAUUGCACUAUC 529 GGGcuAuAAuuGcAcuAucTsT 530GAuAGUGcAAUuAuAGCCCTsT AD- 12312 1104-1122 1509 GAUUCUCUUGGAGGGCGUA 531GAuucucuuGGAGGGcGuATsT 532 uACGCCCUCcAAGAGAAUCTsT AD- 12313 1155-11731510 GCAUCUCUCAAUCUUGAGG 533 GcAucucucAAucuuGAGGTsT 534CCUcAAGAUUGAGAGAUGCTsT AD- 12314 2403-2421 1511 CAGCAGAAAUCUAAGGAUA 535cAGcAGAAAucuAAGGAuATsT 536 uAUCCUuAGAUUUCUGCUGTsT AD- 12315 3115-31331512 GUCAAGAGCCAUCUGUAGA 537 GucAAGAGccAucuGuAGATsT 538UCuAcAGAUGGCUCUUGACTsT AD- 12316 3209-3227 1513 AAACAGAGGCAUUAACACA 539AAAcAGAGGcAuuAAcAcATsT 540 UGUGUuAAUGCCUCUGUUUTsT AD- 12317 3293-33111514 AGCCCAGAUCAACCUUUAA 541 AGcccAGAucAAccuuuAATsT 542UuAAAGGUUGAUCUGGGCUTsT AD- 12318 4574-4592 1515 UAUUUUUGAUCUGGCAACC 543uAuuuuuGAucuGGcAAccTsT 544 GGUUGCcAGAUcAAAAAuATsT AD- 12319 352-370 1516UGUUUGGAGCAUCUACUAA 545 uGuuuGGAGcAucuAcuAATsT 546UuAGuAGAUGCUCcAAAcATsT AD- 12320 741-759 1517 GAAAUUACAGUACACAACA 547GAAAuuAcAGuAcAcAAcATsT 548 UGUUGUGuACUGuAAUUUCTsT AD- 12321 1478-14961518 ACUUGACCAGUGUAAAUCU 549 AcuuGAccAGuGuAAAucuTsT 550AGAUUuAcACUGGUcAAGUTsT AD- 12322 1483-1501 1519 ACCAGUGUAAAUCUGACCU 551AccAGuGuAAAucuGAccuTsT 552 AGGUcAGAUUuAcACUGGUTsT AD- 12323 1967-19851520 AGAACAAUCAUUAGCAGCA 553 AGAAcAAucAuuAGcAGcATsT 554UGCUGCuAAUGAUUGUUCUTsT AD- 12324 2247-2265 1521 CAAUGUGGAAACCUAACUG 555cAAuGuGGAAAccuAAcuGTsT 556 cAGUuAGGUUUCcAcAUUGTsT AD- 12325 2500-25181522 ACCAAGAAGGUACAAAAUU 557 AccAAGAAGGuAcAAAAuuTsT 558AAUUUUGuACCUUCUUGGUTsT AD- 12326 2508-2526 1523 GGUACAAAAUUGGUUGAAG 559GGuAcAAAAuuGGuuGAAGTsT 560 CUUcAACcAAUUUUGuACCTsT AD- 12327 3138-31561524 GGUGUGGAUUGUUCAUCAA 561 GGuGuGGAuuGuucAucAATsT 562UUGAUGAAcAAUCcAcACCTsT AD- 12328 4304-4322 1525 AGAGUUCACAAAAAGCCCA 563AGAGuucAcAAAAAGcccATsT 564 UGGGCUUUUUGUGAACUCUTsT AD- 12329 4711-47291526 UGAUAGCUAAAUUAAACCA 565 uGAuAGcuAAAuuAAAccATsT 566UGGUUuAAUUuAGCuAUcATsT AD- 12330 1221-1239 1527 AAUAAGCCUGAAGUGAAUC 567AAuAAGccuGAAGuGAAucTsT 568 GAUUcACUUcAGGCUuAUUTsT AD- 12331 1705-17231528 CAGUUGACCAACACAAUGC 569 cAGuuGAccAAcAcAAuGcTsT 570GcAUUGUGUUGGUcAACUGTsT AD- 12332 3137-3155 1529 UGGUGUGGAUUGUUCAUCA 571uGGuGuGGAuuGuucAucATsT 572 UGAUGAAcAAUCcAcACcATsT AD- 12333 4292-43101530 AUUCACCCUGACAGAGUUC 573 AuucAcccuGAcAGAGuucTsT 574GAACUCUGUcAGGGUGAAUTsT AD- 12334 1829-1847 1531 UAAGACCUUAUUUGGUAAU 575uAAGAccuuAuuuGGuAAuTsT 576 AUuACcAAAuAAGGUCUuATsT AD- 12335 2244-22621532 AAGCAAUGUGGAAACCUAA 577 AAGcAAuGuGGAAAccuAATsT 578UuAGGUUUCcAcAUUGCUUTsT AD- 12336 2888-2906 1533 UCUGAAACUGGAUAUCCCA 579ucuGAAAcuGGAuAucccATsT 580 UGGGAuAUCcAGUUUcAGATsT AD- 12337

TABLE 2B Table 2B 1st single 2nd single dose screen SDs 1st dose screenSDs 2nd @ 50 nM screen @ 25 nM screen 3rd single SDs 3^(rd) screenduplex [% resudual (among [% resudual (among dose screen (among namemRNA] quadruplicates) mRNA] quadruplicates) @ 25 nM quadruplicates)AD-12072 65% 2% 82% 5% AD-12073 84% 1% 61% 6% AD-12074 51% 3% 36% 9%AD-12075 56% 4% 36% 4% AD-12076 21% 4% 13% 3% AD-12077 11% 2% 6% 1%AD-12078 22% 3% 9% 2% AD-12079 22% 10% 15% 7% AD-12080 68% 4% 52% 13%AD-12081 34% 8% 35% 24% AD-12082 20% 2% 92% 5% AD-12083 85% 6% 63% 10%AD-12084 18% 6% 17% 4% AD-12085 13% 4% 12% 4% AD-12086 26% 5% 17% 3%AD-12087 95% 4% 80% 4% AD-12088 29% 6% 29% 2% AD-12089 69% 5% 64% 7%AD-12090 46% 15% 34% 5% AD-12091 16% 6% 17% 3% AD-12092 82% 26% 63% 5%AD-12093 84% 4% 70% 4% AD-12094 46% 3% 34% 1% AD-12095 14% 2% 13% 1%AD-12096 26% 11% 17% 1% AD-12097 23% 2% 21% 1% AD-12098 41% 14% 17% 3%AD-12099 57% 2% 48% 6% AD-12100 101% 11% 98% 8% AD-12101 46% 7% 32% 2%AD-12102 96% 17% 88% 18% AD-12103 19% 5% 20% 2% AD-12104 40% 8% 24% 2%AD-12105 39% 2% 36% 10% AD-12106 87% 6% 79% 19% AD-12107 29% 2% 32% 16%AD-12108 38% 4% 39% 8% AD-12109 49% 3% 44% 10% AD-12110 85% 5% 80% 14%AD-12111 64% 6% 71% 18% AD-12112 48% 4% 41% 5% AD-12113 13% 0% 14% 3%AD-12114 32% 6% 16% 4% AD-12115 8% 4% 7% 5% AD-12116 74% 5% 61% 7%AD-12117 21% 4% 20% 2% AD-12118 44% 4% 42% 6% AD-12119 37% 4% 24% 3%AD-12120 22% 2% 15% 4% AD-12121 32% 1% 22% 2% AD-12122 36% 16% 19% 5%AD-12123 28% 1% 16% AD-12124 28% 2% 16% AD-12125 15% 1% 14% AD-12126 51%22% 27% AD-12127 54% 4% 42% 9% AD-12128 29% 1% 20% 2% AD-12129 22% 3%19% 3% AD-12130 53% 6% 42% 7% AD-12131 28% 5% 22% 3% AD-12132 88% 2% 90%18% AD-12133 34% 2% 26% 6% AD-12134 18% 3% 14% 2% AD-12135 50% 6% 37% 4%AD-12136 42% 19% 22% 2% AD-12137 85% 12% 92% 4% AD-12138 47% 6% 49% 1%AD-12139 80% 5% 72% 4% AD-12140 97% 22% 67% 9% AD-12141 120% 4% 107% 10%AD-12142 55% 8% 33% 4% AD-12143 64% 34% 19% 2% AD-12144 58% 29% 17% 2%AD-12145 27% 8% 18% 2% AD-12146 19% 20% 15% 1% AD-12147 29% 9% 35% 3%AD-12148 30% 3% 56% 5% AD-12149 8% 2% 12% 3% AD-12150 31% 2% 31% 7%AD-12151 9% 5% 14% 2% AD-12152 3% 3% 23% 3% AD-12153 20% 6% 34% 4%AD-12154 24% 7% 44% 3% AD-12155 33% 6% 53% 11% AD-12156 35% 5% 40% 5%AD-12157 8% 3% 23% 4% AD-12158 13% 2% 22% 5% AD-12159 34% 6% 46% 5%AD-12160 19% 3% 31% 4% AD-12161 88% 4% 83% 7% AD-12162 26% 7% 32% 7%AD-12163 55% 9% 40% 3% AD-12164 21% 3% AD-12165 30% 3% 41% 4% AD-121669% 10% 22% 9% AD-12167 26% 3% 30% 2% AD-12168 54% 4% 59% 20% AD-1216941% 4% 51% 16% AD-12170 43% 4% 52% 20% AD-12171 67% 3% 73% 25% AD-1217253% 15% 37% 2% AD-12173 39% 0% 39% 0% AD-12174 41% 5% 27% 0% AD-1217529% 0% 38% 14% AD-12176 43% 2% 56% 25% AD-12177 68% 6% 74% 30% AD-1217841% 4% 41% 6% AD-12179 53% 5% 44% 5% AD-12180 16% 2% 13% 4% AD-12181 19%3% 14% 2% AD-12182 16% 4% 18% 8% AD-12183 26% 3% 19% 4% AD-12184 54% 2%77% 8% AD-12185 8% 1% 9% 1% AD-12186 36% 3% 41% 6% AD-12187 34% 17% 27%1% AD-12188 30% 3% 27% 4% AD-12189 51% 4% 48% 5% AD-12190 33% 2% 26% 4%AD-12191 20% 2% 13% 0% AD-12192 21% 1% 23% 10% AD-12193 64% 8% 98% 6%AD-12194 8% 2% 15% 4% AD-12195 34% 2% 48% 3% AD-12196 34% 2% 51% 3%AD-12197 75% 4% 93% 6% AD-12198 55% 5% 48% 2% AD-12199 102% 6% 118% 9%AD-12200 75% 6% 60% 12% AD-12201 42% 3% 16% 4% AD-12202 29% 4% 9% 3%AD-12203 114% 14% 89% 20% AD-12204 64% 7% 26% 5% AD-12205 66% 12% 35% 4%AD-12206 46% 3% 32% 12% AD-12207 57% 5% 40% 6% AD-12208 30% 8% 10% 5%AD-12209 101% 6% 102% 23% AD-12210 38% 11% 27% 14% AD-12211 16% 6% 10%5% AD-12212 59% 8% 65% 5% AD-12213 24% 9% 12% 2% AD-12214 67% 14% 70%12% AD-12215 29% 13% 13% 4% AD-12216 36% 4% 13% 1% AD-12217 36% 9% 11%2% AD-12218 35% 5% 17% 3% AD-12219 41% 9% 14% 1% AD-12220 37% 5% 23% 3%AD-12221 58% 7% 39% 6% AD-12222 74% 9% 53% 3% AD-12223 74% 10% 67% 7%AD-12224 24% 2% 11% 2% AD-12225 75% 5% 76% 14% AD-12226 45% 8% 40% 3%AD-12227 61% 6% 47% 5% AD-12228 28% 3% 25% 5% AD-12229 54% 13% 37% 6%AD-12230 70% 17% 65% 4% AD-12231 32% 12% 22% 6% AD-12232 30% 3% 17% 2%AD-12233 38% 2% 32% 3% AD-12234 90% 5% 95% 7% AD-12235 57% 7% 46% 3%AD-12236 34% 8% 16% 2% AD-12237 42% 9% 32% 8% AD-12238 42% 6% 34% 6%AD-12239 42% 3% 40% 4% AD-12240 47% 6% 36% 5% AD-12241 69% 5% 70% 8%AD-12242 61% 2% 47% 3% AD-12243 26% 7% 15% 1% AD-12244 25% 6% 15% 1%AD-12245 65% 6% 83% 13% AD-12246 29% 7% 31% 6% AD-12247 57% 13% 50% 3%AD-12248 36% 8% 20% 3% 15% 7% AD-12249 44% 3% 70% 11% 103% 34% AD-1225047% 5% 18% 5% 17% 4% AD-12251 121% 28% 35% 8% 60% 42% AD-12252 94% 19%8% 3% 5% 3% AD-12253 94% 33% 42% 8% 49% 27% AD-12254 101% 58% 70% 5% 80%32% AD-12255 163% 27% 28% 6% 36% 10% AD-12256 112% 62% 18% 3% 9% 4%AD-12257 10% 4% 9% 2% 6% 2% AD-12258 27% 9% 18% 3% 20% 6% AD-12259 20%5% 12% 2% 13% 5% AD-12260 22% 7% 81% 7% 65% 13% AD-12261 122% 11% 66% 7%80% 22% AD-12262 97% 30% 33% 6% 44% 18% AD-12263 177% 57% 85% 11% 84%15% AD-12264 37% 6% 10% 1% 10% 4% AD-12265 40% 8% 17% 1% 20% 10%AD-12266 33% 9% 9% 1% 8% 4% AD-12267 34% 13% 11% 1% 6% 2% AD-12268 34%6% 11% 1% 9% 2% AD-12269 54% 6% 33% 4% 29% 7% AD-12270 52% 5% 29% 4% 27%6% AD-12271 53% 7% 27% 3% 19% 6% AD-12272 85% 15% 57% 7% 51% 16%AD-12273 36% 6% 26% 2% 30% 5% AD-12274 75% 21% 40% 2% 50% 19% AD-1227529% 9% 8% 1% 8% 4% AD-12276 45% 19% 15% 2% 16% 12% AD-12277 58% 17% 32%2% 55% 14% AD-12278 120% 35% 96% 10% 124% 38% AD-12279 47% 29% 17% 1%12% 4% AD-12280 2% 0% 3% 1% AD-12281 2% 0% 5% 2% AD-12282 3% 0% 25% 5%AD-12283 3% 1% 35% 4% AD-12284 5% 2% 49% 8% AD-12285 7% 7% 21% 26%AD-12286 28% 34% 12% 7% AD-12287 40% 21% 51% 23% AD-12288 26% 7% 155%146% AD-12289 43% 21% 220% 131% AD-12290 2% 1% 81% 23% AD-12291 4% 1%70% 3% AD-12292 2% 1% 6% 2% AD-12293 4% 2% 36% 3% AD-12294 10% 6% 38% 3%AD-12295 29% 31% 37% 3% AD-12296 82% 4% 89% 2% AD-12297 75% 3% 65% 2%AD-12298 73% 4% 60% 3% AD-12299 76% 4% 66% 4% AD-12300 36% 4% 15% 1%AD-12301 33% 4% 18% 2% AD-12302 66% 5% 65% 3% AD-12303 35% 6% 17% 2%AD-12304 70% 8% 70% 6% AD-12305 63% 8% 80% 7% AD-12306 23% 6% 20% 3%AD-12307 78% 10% 58% 5% AD-12308 27% 8% 15% 2% AD-12309 58% 11% 42% 3%AD-12310 106% 23% 80% 2% AD-12311 73% 12% 60% 2% AD-12312 39% 3% 36% 3%AD-12313 64% 9% 49% 6% AD-12314 28% 7% 14% 6% AD-12315 31% 7% 13% 2%AD-12316 42% 5% 14% 2% AD-12317 34% 9% 15% 5% AD-12318 46% 4% 28% 4%AD-12319 77% 3% 56% 4% AD-12320 55% 7% 41% 3% AD-12321 21% 3% 10% 2%AD-12322 27% 8% 30% 12% AD-12323 26% 7% 35% 18% AD-12324 27% 8% 27% 14%AD-12325 32% 12% 32% 22% AD-12326 42% 22% 45% 41% AD-12327 36% 14% 37%32% AD-12328 45% 2% 31% 3% AD-12329 61% 4% 34% 3% AD-12330 63% 5% 38% 4%AD-12331 50% 2% 26% 5% AD-12332 80% 4% 51% 7% AD-12333 34% 6% 12% 2%AD-12334 27% 2% 18% 3% AD-12335 84% 6% 60% 7% AD-12336 45% 4% 36% 4%AD-12337 30% 7% 19% 2%

TABLE 3 TABLE 3 single SDs dose 2nd screen @ screen SEQ SEQ 25 nM [%(among ID ID duplex residual quadru- sequence (5′-3′) NO.sequence (5′-3′) NO. name mRNA] plicates) ccAuuAcuAcAGuAGcAcuTsT  582AGUGCuACUGuAGuAAUGGTsT  583 AD-14085  19%  1% AucuGGcAAccAuAuuucuTsT 584 AGAAAuAUGGUUGCcAGAUTsT  585 AD-14086  38%  1%GAuAGcuAAAuuAAAccAATsT  586 UUGGUUuAAUUuAGCuAUCTsT  587 AD-14087  75%10% AGAuAccAuuAcuAcAGuATsT  588 uACUGuAGuAAUGGuAUCUTsT  589 AD-14088 22%  8% GAuuGuucAucAAuuGGcGTsT  590 CGCcAAUUGAUGAAcAAUCTsT  591AD-14089  70% 12% GcuuucuccucGGcucAcuTsT  592 AGuGAGCCGAGGAGAAAGCTsT 593 AD-14090  79% 11% GGAGGAuuGGcuGAcAAGATsT  594UCUUGUcAGCcAAUCCUCCTsT  595 AD-14091  29%  3% uAAuGAAGAGuAuAccuGGTsT 596 CcAGGuAuACUCUUcAUuATsT  597 AD-14092  23%  2%uuucAccAAAccAuuuGuATsT  598 uAcAAAUGGUUUGGUGAAATsT  599 AD-14093  60% 2% cuuAuuAAGGAGuAuAcGGTsT  600 CCGuAuACUCCUuAAuAAGTsT  601 AD-14094 11%  3% GAAAucAGAuGGAcGuAAGTsT  602 CUuACGUCcAUCUGAUUUCTsT  603AD-14095  10%  2% cAGAuGucAGcAuAAGcGATsT  604 UCGCUuAUGCUGAcAUCUGTsT 605 AD-14096  27%  2% AucuAAcccuAGuuGuAucTsT  606GAuAcAACuAGGGUuAGAUTsT  607 AD-14097  45%  6% AAGAGcuuGuuAAAAucGGTsT 608 CCGAUUUuAAcAAGCUCUUTsT  609 AD-14098  50% 10%uuAAGGAGuAuAcGGAGGATsT  610 UCCUCCGuAuACUCCUuAATsT  611 AD-14099  12% 4% uuGcAAuGuAAAuAcGuAuTsT  612 AuACGuAUUuAcAUUGcAATsT  613 AD-14100 49%  7% ucuAAcccuAGuuGuAuccTsT  614 GGAuAcAACuAGGGUuAGATsT  615AD-14101  36%  1% cAuGuAucuuuuucucGAuTsT  616 AUCGAGAAAAAGAuAcAUGTsT 617 AD-14102  49%  3% GAuGucAGcAuAAGcGAuGTsT  618cAUCGCUuAUGCUGAcAUCTsT  619 AD-14103  74%  5% ucccAAcAGGuAcGAcAccTsT 620 GGUGUCGuACCUGUUGGGATsT  621 AD-14104  27%  3%uGcucAcGAuGAGuuuAGuTsT  622 ACuAAACUcAUCGUGAGcATsT  623 AD-14105  34% 4% AGAGcuuGuuAAAAucGGATsT  624 UCCGAUUUuAAcAAGCUCUTsT  625 AD-14106  9%  2% GcGuAcAAGAAcAucuAuATsT  626 uAuAGAUGUUCUUGuACGCTsT  627AD-14107   5%  1% GAGGuuGuAAGccAAuGuuTsT  628 AAcAUUGGCUuAcAACCUCTsT 629 AD-14108  15%  1% AAcAGGuAcGAcAccAcAGTsT  630CUGUGGUGUCGuACCUGUUTsT  631 AD-14109  91%  2% AAcccuAGuuGuAucccucTsT 632 GAGGGAuAcAACuAGGGUUTsT  633 AD-14110  66%  5%GcAuAAGcGAuGGAuAAuATsT  634 uAUuAUCcAUCGCUuAUGCTsT  635 AD-14111  33% 3% AAGcGAuGGAuAAuAccuATsT  636 uAGGuAUuAUCcAUCGCUUTsT  637 AD-14112 51%  3% uGAuccuGuAcGAAAAGAATsT  638 UUCUUUUCGuAcAGGAUcATsT  639AD-14113  22%  3% AAAAcAuuGGccGuucuGGTsT  640 CcAGAACGGCcAAUGUUUUTsT 641 AD-14114 117%  8% cuuGGAGGGcGuAcAAGAATsT  642UUCUUGuACGCCCUCcAAGTsT  643 AD-14115  50%  8% GGcGuAcAAGAAcAucuAuTsT 644 AuAGAUGUUCUUGuACGCCTsT  645 AD-14116  14%  3%AcucuGAGuAcAuuGGAAuTsT  646 AUUCcAAUGuACUcAGAGUTsT  647 AD-14117  12% 4% uuAuuAAGGAGuAuAcGGATsT  648 UCCGuAuACUCCUuAAuAATsT  649 AD-14118 26%  4% uAAGGAGuAuAcGGAGGAGTsT  650 CUCCUCCGuAuACUCCUuATsT  651AD-14119  24%  5% AAAucAAuAGucAAcuAAATsT  652 UUuAGUUGACuAUUGAUUUTsT 653 AD-14120   8%  1% AAucAAuAGucAAcuAAAGTsT  654CUUuAGUUGACuAUUGAUUTsT  655 AD-14121  24%  2% uucucAGuAuAcuGuGuAATsT 656 UuAcAcAGuAuACUGAGAATsT  657 AD-14122  10%  1%uGuGAAAcAcucuGAuAAATsT  658 UUuAUcAGAGUGUUUcAcATsT  659 AD-14123   8% 1% AGAuGuGAAucucuGAAcATsT  660 UGUUcAGAGAUUcAcAUCUTsT  661 AD-14124  9%  2% AGGuuGuAAGccAAuGuuGTsT  662 cAAcAUUGGCUuAcAACCUTsT  663AD-14125 114%  6% uGAGAAAucAGAuGGAcGuTsT  664 ACGUCcAUCUGAUUUCUcATsT 665 AD-14126   9%  1% AGAAAucAGAuGGAcGuAATsT  666UuACGUCcAUCUGAUUUCUTsT  667 AD-14127  57%  6% AuAucccAAcAGGuAcGAcTsT 668 GUCGuACCUGUUGGGAuAUTsT  669 AD-14128 104%  6%cccAAcAGGuAcGAcAccATsT  670 UGGUGUCGuACCUGUUGGGTsT  671 AD-14129  21% 2% AGuAuAcuGAAGAAccucuTsT  672 AGAGGUUCUUcAGuAuACUTsT  673 AD-14130 57%  6% AuAuAuAucAGccGGGcGcTsT  674 GCGCCCGGCUGAuAuAuAUTsT  675AD-14131  93%  6% AAucuAAcccuAGuuGuAuTsT  676 AuAcAACuAGGGUuAGAUUTsT 677 AD-14132  75%  8% cuAAcccuAGuuGuAucccTsT  678GGGAuAcAACuAGGGUuAGTsT  679 AD-14133  66%  4% cuAGuuGuAucccuccuuuTsT 680 AAAGGAGGGAuAcAACuAGTsT  681 AD-14134  44%  6%AGAcAucuGAcuAAuGGcuTsT  682 AGCcAUuAGUcAGAUGUCUTsT  683 AD-14135  55% 6% GAAGcucAcAAuGAuuuAATsT  684 UuAAAUcAUUGUGAGCUUCTsT  685 AD-14136 29%  3% AcAuGuAucuuuuucucGATsT  686 UCGAGAAAAAGAuAcAUGUTsT  687AD-14137  40%  3% ucGAuucAAAucuuAAcccTsT  688 GGGUuAAGAUUUGAAUCGATsT 689 AD-14138  39%  5% ucuuAAcccuuAGGAcucuTsT  690AGAGUCCuAAGGGUuAAGATsT  691 AD-14139  71% 11% GcucAcGAuGAGuuuAGuGTsT 692 cACuAAACUcAUCGUGAGCTsT  693 AD-14140  43% 15%cAuAAGcGAuGGAuAAuAcTsT  694 GuAUuAUCcAUCGCUuAUGTsT  695 AD-14141  33% 6% AuAAGcGAuGGAuAAuAccTsT  696 GGuAUuAUCcAUCGCUuAUTsT  697 AD-14142 51% 14% ccuAAuAAAcuGcccucAGTsT  698 CUGAGGGcAGUUuAUuAGGTsT  699AD-14143  42%  1% ucGGAAAGuuGAAcuuGGuTsT  700 ACcAAGUUcAACUUUCCGATsT 701 AD-14144   4%  4% GAAAAcAuuGGccGuucuGTsT  702cAGAACGGCcAAUGUUUUCTsT  703 AD-14145  92%  5% AAGAcuGAucuucuAAGuuTsT 704 AACUuAGAAGAUcAGUCUUTsT  705 AD-14146  13%  2%GAGcuuGuuAAAAucGGAATsT  706 UUCCGAUUUuAAcAAGCUCTsT  707 AD-14147   8% 1% AcAuuGGccGuucuGGAGcTsT  708 GCUCcAGAACGGCcAAUGUTsT  709 AD-14148 80%  7% AAGAAcAucuAuAAuuGcATsT  710 UGcAAUuAuAGAUGUUCUUTsT  711AD-14149  44%  7% AAAuGuGucuAcucAuGuuTsT  712 AAcAUGAGuAGAcAcAUUUTsT 713 AD-14150  32% 29% uGucuAcucAuGuuucucATsT  714UGAGAAAcAUGAGuAGAcATsT  715 AD-14151  75% 11% GuAuAcuGuGuAAcAAucuTsT 716 AGAUUGUuAcAcAGuAuACTsT  717 AD-14152   8%  5%uAuAcuGuGuAAcAAucuATsT  718 uAGAUUGUuAcAcAGuAuATsT  719 AD-14153  17%11% cuuAGuAGuGuccAGGAAATsT  720 UUUCCUGGAcACuACuAAGTsT  721 AD-14154 16%  4% ucAGAuGGAcGuAAGGcAGTsT  722 CUGCCUuACGUCcAUCUGATsT  723AD-14155  11%  1% AGAuAAAuuGAuAGcAcAATsT  724 UUGUGCuAUcAAUUuAUCUTsT 725 AD-14156  10%  1% cAAcAGGuAcGAcAccAcATsT  726UGUGGUGUCGuACCUGUUGTsT  727 AD-14157  29%  3% uGcAAuGuAAAuAcGuAuuTsT 728 AAuACGuAUUuAcAUUGcATsT  729 AD-14158  51%  3%AGucAGAAuuuuAucuAGATsT  730 UCuAGAuAAAAUUCUGACUTsT  731 AD-14159  53% 5% cuAGAAAucuuuuAAcAccTsT  732 GGUGUuAAAAGAUUUCuAGTsT  733 AD-14160 40%  3% AAuAAAucuAAcccuAGuuTsT  734 AACuAGGGUuAGAUUuAUUTsT  735AD-14161  83%  7% AAuuuucuGcucAcGAuGATsT  736 UcAUCGUGAGcAGAAAAUUTsT 737 AD-14162  44%  6% GcccucAGuAAAuccAuGGTsT  738CcAUGGAUUuACUGAGGGCTsT  739 AD-14163  57%  3% AcGuuuAAAAcGAGAucuuTsT 740 AAGAUCUCGUUUuAAACGUTsT  741 AD-14164   4%  1%AGGAGAuAGAAcGuuuAAATsT  742 UUuAAACGUUCuAUCUCCUTsT  743 AD-14165  11% 1% GAccGucAuGGcGucGcAGTsT  744 CUGCGACGCcAUGACGGUCTsT  745 AD-14166 90%  5% AccGucAuGGcGucGcAGcTsT  746 GCUGCGACGCcAUGACGGUTsT  747AD-14167  49%  1% GAAcGuuuAAAAcGAGAucTsT  748 GAUCUCGUUUuAAACGUUCTsT 749 AD-14168  12%  2% uuGAGcuuAAcAuAGGuAATsT  750UuACCuAUGUuAAGCUcAATsT  751 AD-14169  66%  4% AcuAAAuuGAucucGuAGATsT 752 UCuACGAGAUcAAUUuAGUTsT  753 AD-14170  52%  6%ucGuAGAAuuAucuuAAuATsT  754 uAUuAAGAuAAUUCuACGATsT  755 AD-14171  42% 4% GGAGAuAGAAcGuuuAAAATsT  756 UUUuAAACGUUCuAUCUCCTsT  757 AD-14172  3%  1% AcAAcuuAuuGGAGGuuGuTsT  758 AcAACCUCcAAuAAGUUGUTsT  759AD-14173  29%  2% uGAGcuuAAcAuAGGuAAATsT  760 UUuACCuAUGUuAAGCUcATsT 761 AD-14174  69%  2% AucucGuAGAAuuAucuuATsT  762uAAGAuAAUUCuACGAGAUTsT  763 AD-14175  53%  3% cuGcGuGcAGucGGuccucTsT 764 GAGGACCGACUGcACGcAGTsT  765 AD-14176 111%  4%cAcGcAGcGcccGAGAGuATsT  766 uACUCUCGGGCGCUGCGUGTsT  767 AD-14177  87% 6% AGuAccAGGGAGAcuccGGTsT  768 CCGGAGUCUCCCUGGuACUTsT  769 AD-14178 59%  2% AcGGAGGAGAuAGAAcGuuTsT  770 AACGUUCuAUCUCCUCCGUTsT  771AD-14179   9%  2% AGAAcGuuuAAAAcGAGAuTsT  772 AUCUCGUUUuAAACGUUCUTsT 773 AD-14180  43%  2% AAcGuuuAAAAcGAGAucuTsT  774AGAUCUCGUUUuAAACGUUTsT  775 AD-14181  70% 10% AGcuuGAGcuuAAcAuAGGTsT 776 CCuAUGUuAAGCUcAAGCUTsT  777 AD-14182 100%  7%AGcuuAAcAuAGGuAAAuATsT  778 uAUUuACCuAUGUuAAGCUTsT  779 AD-14183  60% 5% uAGAGcuAcAAAAccuAucTsT  780 GAuAGGUUUUGuAGCUCuATsT  781 AD-14184129%  6% uAGuuGuAucccuccuuuATsT  782 uAAAGGAGGGAuAcAACuATsT  783AD-14185  62%  4% AccAcccAGAcAucuGAcuTsT  784 AGUcAGAUGUCUGGGUGGUTsT 785 AD-14186  42%  3% AGAAAcuAAAuuGAucucGTsT  786CGAGAUcAAUUuAGUUUCUTsT  787 AD-14187 123% 12% ucucGuAGAAuuAucuuAATsT 788 UuAAGAuAAUUCuACGAGATsT  789 AD-14188  38%  2%cAAcuuAuuGGAGGuuGuATsT  790 uAcAACCUCcAAuAAGUUGTsT  791 AD-14189  13% 1% uuGuAucccuccuuuAAGuTsT  792 ACUuAAAGGAGGGAuAcAATsT  793 AD-14190 59%  3% ucAcAAcuuAuuGGAGGuuTsT  794 AACCUCcAAuAAGUUGUGATsT  795AD-14191  93%  3% AGAAcuGuAcucuucucAGTsT  796 CUGAGAAGAGuAcAGUUCUTsT 797 AD-14192  45%  5% GAGcuuAAcAuAGGuAAAuTsT  798AUUuACCuAUGUuAAGCUCTsT  799 AD-14193  57%  3% cAccAAcAucuGuccuuAGTsT 800 CuAAGGAcAGAUGUUGGUGTsT  801 AD-14194  51%  4%AAAGcccAcuuuAGAGuAuTsT  802 AuACUCuAAAGUGGGCUUUTsT  803 AD-14195  77% 5% AAGcccAcuuuAGAGuAuATsT  804 uAuACUCuAAAGUGGGCUUTsT  805 AD-14196 42%  6% GAccuuAuuuGGuAAucuGTsT  806 cAGAUuACcAAAuAAGGUCTsT  807AD-14197  15%  2% GAuuAAuGuAcucAAGAcuTsT  808 AGUCUUGAGuAcAUuAAUCTsT 809 AD-14198  12%  2% cuuuAAGAGGccuAAcucATsT  810UGAGUuAGGCCUCUuAAAGTsT  811 AD-14199  18%  2% uuAAAccAAAcccuAuuGATsT 812 UcAAuAGGGUUUGGUUuAATsT  813 AD-14200  72%  9%ucuGuuGGAGAucuAuAAuTsT  814 AUuAuAGAUCUCcAAcAGATsT  815 AD-14201   9% 3% cuGAuGuuucuGAGAGAcuTsT  816 AGUCUCUcAGAAAcAUcAGTsT  817 AD-14202 25%  3% GcAuAcucuAGucGuucccTsT  818 GGGAACGACuAGAGuAUGCTsT  819AD-14203  21%  1% GuuccuuAucGAGAAucuATsT  820 uAGAUUCUCGAuAAGGAACTsT 821 AD-14204   4%  2% GcAcuuGGAucucucAcAuTsT  822AUGUGAGAGAUCcAAGUGCTsT  823 AD-14205   5%  1% AAAAAAGGAAcuAGAuGGcTsT 824 GCcAUCuAGUUCCUUUUUUTsT  825 AD-14206  79%  6%AGAGcAGAuuAccucuGcGTsT  826 CGcAGAGGuAAUCUGCUCUTsT  827 AD-14207  55% 2% AGcAGAuuAccucuGcGAGTsT  828 CUCGcAGAGGuAAUCUGCUTsT  829 AD-14208100%  4% cccuGAcAGAGuucAcAAATsT  830 UUUGUGAACUCUGUcAGGGTsT  831AD-14209  34%  3% GuuuAccGAAGuGuuGuuuTsT  832 AAAcAAcACUUCGGuAAACTsT 833 AD-14210  13%  2% uuAcAGuAcAcAAcAAGGATsT  834UCCUUGUUGUGuACUGuAATsT  835 AD-14211   9%  1% AcuGGAucGuAAGAAGGcATsT 836 UGCCUUCUuACGAUCcAGUTsT  837 AD-14212  20%  3%GAGcAGAuuAccucuGcGATsT  838 UCGcAGAGGuAAUCUGCUCTsT  839 AD-14213  48% 5% AAAAGAAGuuAGuGuAcGATsT  840 UCGuAcACuAACUUCUUUUTsT  841 AD-14214 28% 18% GAccAuuuAAuuuGGcAGATsT  842 UCUGCcAAAUuAAAUGGUCTsT  843AD-14215 132%  0% GAGAGGAGuGAuAAuuAAATsT  844 UUuAAUuAUcACUCCUCUCTsT 845 AD-14216   3%  0% cuGGAGGAuuGGcuGAcAATsT  846UUGUcAGCcAAUCCUCcAGTsT  847 AD-14217  19%  1% cucuAGucGuucccAcucATsT 848 UGAGUGGGAACGACuAGAGTsT  849 AD-14218  67%  8%GAuAccAuuAcuAcAGuAGTsT  850 CuACUGuAGuAAUGGuAUCTsT  851 AD-14219  76% 4% uucGucuGcGAAGAAGAAATsT  852 UUUCUUCUUCGcAGACGAATsT  853 AD-14220 33%  8% GAAAAGAAGuuAGuGuAcGTsT  854 CGuAcACuAACUUCUUUUCTsT  855AD-14221  25%  2% uGAuGuuuAccGAAGuGuuTsT  856 AAcACUUCGGuAAAcAUcATsT 857 AD-14222   7%  2% uGuuuGuccAAuucuGGAuTsT  858AUCcAGAAUUGGAcAAAcATsT  859 AD-14223  19%  2% AuGAAGAGuAuAccuGGGATsT 860 UCCcAGGuAuACUCUUcAUTsT  861 AD-14224  13%  1%GcuAcucuGAuGAAuGcAuTsT  862 AUGcAUUcAUcAGAGuAGCTsT  863 AD-14225  15% 2% GcccuuGuAGAAAGAAcAcTsT  864 GUGUUCUUUCuAcAAGGGCTsT  865 AD-14226 11%  0% ucAuGuuccuuAucGAGAATsT  866 UUCUCGAuAAGGAAcAUGATsT  867AD-14227   5%  1% GAAuAGGGuuAcAGAGuuGTsT  868 cAACUCUGuAACCCuAUUCTsT 869 AD-14228  34%  3% cAAAcuGGAucGuAAGAAGTsT  870CUUCUuACGAUCcAGUUUGTsT  871 AD-14229  15%  2% cuuAuuuGGuAAucuGcuGTsT 872 cAGcAGAUuACcAAAuAAGTsT  873 AD-14230  20%  1%AGcAAuGuGGAAAccuAAcTsT  874 GUuAGGUUUCcAcAUUGCUTsT  875 AD-14231  18% 1% AcAAuAAAGcAGAcccAuuTsT  876 AAUGGGUCUGCUUuAUUGUTsT  877 AD-14232 21%  1% AAccAcuuAGuAGuGuccATsT  878 UGGAcACuACuAAGUGGUUTsT  879AD-14233 106% 12% AGucAAGAGccAucuGuAGTsT  880 CuAcAGAUGGCUCUUGACUTsT 881 AD-14234  35%  3% cucccuAGAcuucccuAuuTsT  882AAuAGGGAAGUCuAGGGAGTsT  883 AD-14235  48%  4% AuAGcuAAAuuAAAccAAATsT 884 UUUGGUUuAAUUuAGCuAUTsT  885 AD-14236  23%  3%uGGcuGGuAuAAuuccAcGTsT  886 CGUGGAAUuAuACcAGCcATsT  887 AD-14237  79% 9% uuAuuuGGuAAucuGcuGuTsT  888 AcAGcAGAUuACcAAAuAATsT  889 AD-14238 92%  7% AAcuAGAuGGcuuucucAGTsT  890 CUGAGAAAGCcAUCuAGUUTsT  891AD-14239  20%  2% ucAuGGcGucGcAGccAAATsT  892 UUUGGCUGCGACGCcAUGATsT 893 AD-14240  71%  6% AcuGGAGGAuuGGcuGAcATsT  894UGUcAGCcAAUCCUCcAGUTsT  895 AD-14241  14%  1% cuAuAAuuGcAcuAucuuuTsT 896 AAAGAuAGUGcAAUuAuAGTsT  897 AD-14242  11%  2%AAAGGucAccuAAuGAAGATsT  898 UCUUcAUuAGGUGACCUUUTsT  899 AD-14243  11% 1% AuGAAuGcAuAcucuAGucTsT  900 GACuAGAGuAUGcAUUcAUTsT  901 AD-14244 15%  2% AAcAuAuuGAAuAAGccuGTsT  902 cAGGCUuAUUcAAuAUGUUTsT  903AD-14245  80%  7% AAGAAGGcAGuuGAccAAcTsT  904 GUUGGUcAACUGCCUUCUUTsT 905 AD-14246  57%  5% GAuAcuAAAAGAAcAAucATsT  906UGAUUGUUCUUUuAGuAUCTsT  907 AD-14247   9%  3% AuAcuGAAAAucAAuAGucTsT 908 GACuAUUGAUUUUcAGuAUTsT  909 AD-14248  39%  4%AAAAAGGAAcuAGAuGGcuTsT  910 AGCcAUCuAGUUCCUUUUUTsT  911 AD-14249  64% 2% GAAcuAGAuGGcuuucucATsT  912 UGAGAAAGCcAUCuAGUUCTsT  913 AD-14250 18%  2% GAAAccuAAcuGAAGAccuTsT  914 AGGUCUUcAGUuAGGUUUCTsT  915AD-14251  56%  6% uAcccAucAAcAcuGGuAATsT  916 UuACcAGUGUUGAUGGGuATsT 917 AD-14252  48%  6% AuuuuGAuAucuAcccAuuTsT  918AAUGGGuAGAuAUcAAAAUTsT  919 AD-14253  39%  5% AucccuAuAGuucAcuuuGTsT 920 cAAAGUGAACuAuAGGGAUTsT  921 AD-14254  44%  8%AuGGGcuAuAAuuGcAcuATsT  922 uAGUGcAAUuAuAGCCcAUTsT  923 AD-14255 108% 8% AGAuuAccucuGcGAGcccTsT  924 GGGCUCGcAGAGGuAAUCUTsT  925 AD-14256108%  6% uAAuuccAcGuAcccuucATsT  926 UGAAGGGuACGUGGAAUuATsT  927AD-14257  23%  2% GucGuucccAcucAGuuuuTsT  928 AAAACuGAGuGGGAACGACTsT 929 AD-14258  21%  3% AAAucAAucccuGuuGAcuTsT  930AGUcAAcAGGGAUUGAUUUTsT  931 AD-14259  19%  2% ucAuAGAGcAAAGAAcAuATsT 932 uAUGUUCUUUGCUCuAUGATsT  933 AD-14260  10%  1%uuAcuAcAGuAGcAcuuGGTsT  934 CcAAGUGCuACUGuAGuAATsT  935 AD-14261  76% 3% AuGuGGAAAccuAAcuGAATsT  936 UUcAGUuAGGUUUCcAcAUTsT  937 AD-14262 13%  2% uGuGGAAAccuAAcuGAAGTsT  938 CUUcAGUuAGGUUUCcAcATsT  939AD-14263  14%  2% ucuuccuuAAAuGAAAGGGTsT  940 CCCUUUcAUUuAAGGAAGATsT 941 AD-14264  65%  3% uGAAGAAccucuAAGucAATsT  942UUGACUuAGAGGUUCUUcATsT  943 AD-14265  13%  1% AGAGGucuAAAGuGGAAGATsT 944 UCUUCcACUUuAGACCUCUTsT  945 AD-14266  18%  3%AuAucuAcccAuuuuucuGTsT  946 cAGAAAAAUGGGuAGAuAUTsT  947 AD-14267  50% 9% uAAGccuGAAGuGAAucAGTsT  948 CUGAUUcACUUcAGGCUuATsT  949 AD-14268 13%  3% AGAuGcAGAccAuuuAAuuTsT  950 AAUuAAAUGGUCUGcAUCUTsT  951AD-14269  19%  4% AGuGuuGuuuGuccAAuucTsT  952 GAAUUGGAcAAAcAAcACUTsT 953 AD-14270  11%  2% cuAuAAuGAAGAGcuuuuuTsT  954AAAAAGCUCUUcAUuAuAGTsT  955 AD-14271  11%  1% AGAGGAGuGAuAAuuAAAGTsT 956 CUUuAAUuAUcACUCCUCUTsT  957 AD-14272   7%  1%uuucucuGuuAcAAuAcAuTsT  958 AUGuAUUGuAAcAGAGAAATsT  959 AD-14273  14% 2% AAcAucuAuAAuuGcAAcATsT  960 UGUUGcAAUuAuAGAUGUUTsT  961 AD-14274 73%  4% uGcuAGAAGuAcAuAAGAcTsT  962 GUCUuAUGuACUUCuAGcATsT  963AD-14275  10%  1% AAuGuAcucAAGAcuGAucTsT  964 GAUcAGUCUUGAGuAcAUUTsT 965 AD-14276  89%  2% GuAcucAAGAcuGAucuucTsT  966GAAGAUcAGUCUUGAGuACTsT  967 AD-14277   7%  1% cAcucuGAuAAAcucAAuGTsT 968 cAUUGAGUUuAUcAGAGUGTsT  969 AD-14278  12%  1%AAGAGcAGAuuAccucuGcTsT  970 GcAGAGGuAAUCUGCUCUUTsT  971 AD-14279 104% 3% ucuGcGAGcccAGAucAAcTsT  972 GUUGAUCUGGGCUCGcAGATsT  973 AD-14280 21%  2% AAcuuGAGccuuGuGuAuATsT  974 uAuAcAcAAGGCUcAAGUUTsT  975AD-14281  43%  3% GAAuAuAuAuAucAGccGGTsT  976 CCGGCUGAuAuAuAuAUUCTsT 977 AD-14282  45%  6% uGucAucccuAuAGuucAcTsT  978GUGAACuAuAGGGAUGAcATsT  979 AD-14283  35%  5% GAucuGGcAAccAuAuuucTsT 980 GAAAuAUGGUUGCcAGAUCTsT  981 AD-14284  58%  3%uGGcAAccAuAuuucuGGATsT  982 UCcAGAAAuAUGGUUGCcATsT  983 AD-14285  48% 3% GAuGuuuAccGAAGuGuuGTsT  984 cAAcACUUCGGuAAAcAUCTsT  985 AD-14286 49%  3% uuccuuAucGAGAAucuAATsT  986 UuAGAUUCUCGAuAAGGAATsT  987AD-14287   6%  1% AGcuuAAuuGcuuucuGGATsT  988 UCcAGAAAGcAAUuAAGCUTsT 989 AD-14288  50%  2% uuGcuAuuAuGGGAGAccATsT  990UGGUCUCCcAuAAuAGcAATsT  991 AD-14289  48%  1% GucAuGGcGucGcAGccAATsT 992 UUGGCUGCGACGCcAUGACTsT  993 AD-14290 112%  7%uAAuuGcAcuAucuuuGcGTsT  994 CGcAAAGAuAGUGcAAUuATsT  995 AD-14291  77% 2% cuAucuuuGcGuAuGGccATsT  996 UGGCcAuACGcAAAGAuAGTsT  997 AD-14292 80%  6% ucccuAuAGuucAcuuuGuTsT  998 AcAAAGUGAACuAuAGGGATsT  999AD-14293  58%  2% ucAAccuuuAAuucAcuuGTsT 1000 cAAGUGAAUuAAAGGUUGATsT1001 AD-14294  77%  2% GGcAAccAuAuuucuGGAATsT 1002UUCcAGAAAuAUGGUUGCCTsT 1003 AD-14295  62%  2% AuGuAcucAAGAcuGAucuTsT1004 AGAUcAGUCUUGAGuAcAUTsT 1005 AD-14296  59%  4%GcAGAccAuuuAAuuuGGcTsT 1006 GCcAAAUuAAAUGGUCUGCTsT 1007 AD-14297  37% 1% ucuGAGAGAcuAcAGAuGuTsT 1008 AcAUCUGuAGUCUCUcAGATsT 1009 AD-14298 21%  1% uGcucAuAGAGcAAAGAAcTsT 1010 GUUCUUUGCUCuAUGAGcATsT 1011AD-14299   6%  1% AcAuAAGAccuuAuuuGGuTsT 1012 ACcAAAuAAGGUCUuAUGUTsT1013 AD-14300  17%  2% uuuGuGcuGAuucuGAuGGTsT 1014CcAUcAGAAUcAGcAcAAATsT 1015 AD-14301  97%  6% ccAucAAcAcuGGuAAGAATsT1016 UUCUuACcAGUGUUGAUGGTsT 1017 AD-14302  13%  1%AGAcAAuuccGGAuGuGGATsT 1018 UCcAcAUCCGGAAUUGUCUTsT 1019 AD-14303  13% 3% GAAcuuGAGccuuGuGuAuTsT 1020 AuAcAcAAGGCUcAAGUUCTsT 1021 AD-14304 38%  2% uAAuuuGGcAGAGcGGAAATsT 1022 UUUCCGCUCUGCcAAAUuATsT 1023AD-14305  14%  2% uGGAuGAAGuuAuuAuGGGTsT 1024 CCcAuAAuAACUUcAUCcATsT1025 AD-14306  22%  4% AucuAcAuGAAcuAcAAGATsT 1026UCUUGuAGUUcAUGuAGAUTsT 1027 AD-14307  26%  6% GGuAuuuuuGAucuGGcAATsT1028 UUGCcAGAUcAAAAAuACCTsT 1029 AD-14308  62%  8%cuAAuGAAGAGuAuAccuGTsT 1030 cAGGuAuACUCUUcAUuAGTsT 1031 AD-14309  52% 5% uuuGAGAAAcuuAcuGAuATsT 1032 uAUcAGuAAGUUUCUcAAATsT 1033 AD-14310 32%  3% cGAuAAGAuAGAAGAucAATsT 1034 UUGAUCUUCuAUCUuAUCGTsT 1035AD-14311  23%  2% cuGGcAAccAuAuuucuGGTsT 1036 CcAGAAAuAUGGUUGCcAGTsT1037 AD-14312  49%  6% uAGAuAccAuuAcuAcAGuTsT 1038ACUGuAGuAAUGGuAUCuATsT 1039 AD-14313  69%  4% GuAuuAAAuuGGGuuucAuTsT1040 AUGAAACCcAAUUuAAuACTsT 1041 AD-14314  52%  3%AAGAccuuAuuuGGuAAucTsT 1042 GAUuACcAAAuAAGGUCUUTsT 1043 AD-14315  66% 4% GcuGuuGAuAAGAGAGcucTsT 1044 GAGCUCUCUuAUcAAcAGCTsT 1045 AD-14316 19%  4% uAcucAuGuuucucAGAuuTsT 1046 AAUCUGAGAAAcAUGAGuATsT 1047AD-14317  16%  5% cAGAuGGAcGuAAGGcAGcTsT 1048 GCUGCCUuACGUCcAUCUGTsT1049 AD-14318  52% 11% uAucccAAcAGGuAcGAcATsT 1050UGUCGuACCUGUUGGGAuATsT 1051 AD-14319  28% 11% cAuuGcuAuuAuGGGAGAcTsT1052 GUCUCCcAuAAuAGcAAUGTsT 1053 AD-14320  52% 10%cccucAGuAAAuccAuGGuTsT 1054 ACcAUGGAUUuACUGAGGGTsT 1055 AD-14321  53% 6% GGucAuuAcuGcccuuGuATsT 1056 uAcAAGGGcAGuAAUGACCTsT 1057 AD-14322 20%  2% AAccAcucAAAAAcAuuuGTsT 1058 cAAAUGUUUUUGAGUGGUUTsT 1059AD-14323 116%  6% uuuGcAAGuuAAuGAAucuTsT 1060 AGAUUcAUuAACUUGcAAATsT1061 AD-14324  14%  2% uuAuuuucAGuAGucAGAATsT 1062UUCUGACuACUGAAAAuAATsT 1063 AD-14325  50%  2% uuuucucGAuucAAAucuuTsT1064 AAGAUUuGAAUCGAGAAAATsT 1065 AD-14326  47%  3%GuAcGAAAAGAAGuuAGuGTsT 1066 cACuAACUUCUUUUCGuACTsT 1067 AD-14327  18% 2% uuuAAAAcGAGAucuuGcuTsT 1068 AGcAAGAUCUCGUUUuAAATsT 1069 AD-14328 19%  1% GAAuuGAuuAAuGuAcucATsT 1070 UGAGuAcAUuAAUcAAUUCTsT 1071AD-14329  94% 10% GAuGGAcGuAAGGcAGcucTsT 1072 GAGCUGCCUuACGUCcAUCTsT1073 AD-14330  60%  4% cAucuGAcuAAuGGcucuGTsT 1074cAGAGCcAUuAGUcAGAUGTsT 1075 AD-14331  54%  7% GuGAuccuGuAcGAAAAGATsT1076 UCUUUUCGuAcAGGAUcACTsT 1077 AD-14332  22%  4%AGcucuuAuuAAGGAGuAuTsT 1078 AuACUCCUuAAuAAGAGCUTsT 1079 AD-14333  70%10% GcucuuAuuAAGGAGuAuATsT 1080 uAuACUCCUuAAuAAGAGCTsT 1081 AD-14334 18%  3% ucuuAuuAAGGAGuAuAcGTsT 1082 CGuAuACUCCUuAAuAAGATsT 1083AD-14335  38%  6% uAuuAAGGAGuAuAcGGAGTsT 1084 CUCCGuAuACUCCUuAAuATsT1085 AD-14336  16%  3% cuGcAGcccGuGAGAAAAATsT 1086UUUUUCUcACGGGCUGcAGTsT 1087 AD-14337  65%  4% ucAAGAcuGAucuucuAAGTsT1088 CUuAGAAGAUcAGUCUUGATsT 1089 AD-14338  18%  0%cuucuAAGuucAcuGGAAATsT 1090 UUUCcAGUGAACUuAGAAGTsT 1091 AD-14339  20% 4% uGcAAGuuAAuGAAucuuuTsT 1092 AAAGAUUcAUuAACUUGcATsT 1093 AD-14340 24%  1% AAucuAAGGAuAuAGucAATsT 1094 UUGACuAuAUCCUuAGAUUTsT 1095AD-14341  27%  3% AucucuGAAcAcAAGAAcATsT 1096 UGUUCUUGUGUUcAGAGAUTsT1097 AD-14342  13%  1% uucuGAAcAGuGGGuAucuTsT 1098AGAuACCcACUGUUcAGAATsT 1099 AD-14343  19%  1% AGuuAuuuAuAcccAucAATsT1100 UUGAUGGGuAuAAAuAACUTsT 1101 AD-14344  23%  2%AuGcuAAAcuGuucAGAAATsT 1102 UUUCUGAAcAGUUuAGcAUTsT 1103 AD-14345  21% 4% cuAcAGAGcAcuuGGuuAcTsT 1104 GuAACcAAGUGCUCUGuAGTsT 1105 AD-14346 18%  2% uAuAuAucAGccGGGcGcGTsT 1106 CGCGCCCGGCUGAuAuAuATsT 1107AD-14347  67%  2% AuGuAAAuAcGuAuuucuATsT 1108 uAGAAAuACGuAUUuAcAUTsT1109 AD-14348  39%  3% uuuuucucGAuucAAAucuTsT 1110AGAUUuGAAUCGAGAAAAATsT 1111 AD-14349  83%  6% AAucuuAAcccuuAGGAcuTsT1112 AGUCCuAAGGGUuAAGAUUTsT 1113 AD-14350  54%  2%ccuuAGGAcucuGGuAuuuTsT 1114 AAAuACcAGAGUCCuAAGGTsT 1115 AD-14351  57% 8% AAuAAAcuGcccucAGuAATsT 1116 UuACUGAGGGcAGUUuAUUTsT 1117 AD-14352 82%  3% GAuccuGuAcGAAAAGAAGTsT 1118 CUUCUUUUCGuAcAGGAUCTsT 1119AD-14353   2%  1% AAuGuGAuccuGuAcGAAATsT 1120 UUUCGuAcAGGAUcAcAUUTsT1121 AD-14354  18% 11% GuGAAAAcAuuGGccGuucTsT 1122GAACGGCcAAUGUUUUcACTsT 1123 AD-14355   2%  1% cuuGAGGAAAcucuGAGuATsT1124 uACUcAGAGUUUCCUcAAGTsT 1125 AD-14356   8%  2%cGuuuAAAAcGAGAucuuGTsT 1126 cAAGAUCUCGUUUuAAACGTsT 1127 AD-14357   6% 3% uuAAAAcGAGAucuuGcuGTsT 1128 cAGcAAGAUCUCGUUUuAATsT 1129 AD-14358 98% 17% AAAGAuGuAucuGGucuccTsT 1130 GGAGACcAGAuAcAUCUUUTsT 1131AD-14359  10%  1% cAGAAAAuGuGucuAcucATsT 1132 UGAGuAGAcAcAUUUUCUGTsT1133 AD-14360   6%  4% cAGGAAuuGAuuAAuGuAcTsT 1134GuAcAUuAAUcAAUUCCUGTsT 1135 AD-14361  30%  5% AGucAAcuAAAGcAuAuuuTsT1136 AAAuAUGCUUuAGUUGACUTsT 1137 AD-14362  28%  2%uGuGuAAcAAucuAcAuGATsT 1138 UcAUGuAGAUUGUuAcAcATsT 1139 AD-14363  60% 6% AuAccAuuuGuuccuuGGuTsT 1140 ACcAAGGAAcAAAUGGuAUTsT 1141 AD-14364 12%  9% GcAGAAAucuAAGGAuAuATsT 1142 uAuAUCCUuAGAUUUCUGCTsT 1143AD-14365   5%  2% uGGcuucucAcAGGAAcucTsT 1144 GAGUUCCUGUGAGAAGCcATsT1145 AD-14366  28%  5% GAGAuGuGAAucucuGAAcTsT 1146GUUcAGAGAUUcAcAUCUCTsT 1147 AD-14367  42%  4% uGuAAGccAAuGuuGuGAGTsT1148 CUcAcAAcAUUGGCUuAcATsT 1149 AD-14368  93% 12%AGccAAuGuuGuGAGGcuuTsT 1150 AAGCCUcAcAAcAUUGGCUTsT 1151 AD-14369  65% 4% uuGuGAGGcuucAAGuucATsT 1152 UGAACUUGAAGCCUcAcAATsT 1153 AD-14370  5%  2% AGGcAGcucAuGAGAAAcATsT 1154 UGUUUCUcAUGAGCUGCCUTsT 1155AD-14371  54%  5% AuAAAuuGAuAGcAcAAAATsT 1156 UUUUGUGCuAUcAAUUuAUTsT1157 AD-14372   4%  1% AcAAAAucuAGAAcuuAAuTsT 1158AUuAAGUUCuAGAUUUUGUTsT 1159 AD-14373   5%  1% GAuAucccAAcAGGuAcGATsT1160 UCGuACCUGUUGGGAuAUCTsT 1161 AD-14374  92%  6%AAGuuAuuuAuAcccAucATsT 1162 UGAUGGGuAuAAAuAACUUTsT 1163 AD-14375  76% 4% uGuAAAuAcGuAuuucuAGTsT 1164 CuAGAAAuACGuAUUuAcATsT 1165 AD-14376 70%  5% ucuAGuuuucAuAuAAAGuTsT 1166 ACUUuAuAUGAAAACuAGATsT 1167AD-14377  48%  4% AuAAAGuAGuucuuuuAuATsT 1168 uAuAAAAGAACuACUUuAUTsT1169 AD-14378  48%  3% ccAuuuGuAGAGcuAcAAATsT 1170UUUGuAGCUCuAcAAAUGGTsT 1171 AD-14379  44%  5% uAuuuucAGuAGucAGAAuTsT1172 AUUCUGACuACUGAAAAuATsT 1173 AD-14380  35% 16%AAAucuAAcccuAGuuGuATsT 1174 uAcAACuAGGGUuAGAUUUTsT 1175 AD-14381  44% 5% cuuuAGAGuAuAcAuuGcuTsT 1176 AGcAAUGuAuACUCuAAAGTsT 1177 AD-14382 28%  1% AucuGAcuAAuGGcucuGuTsT 1178 AcAGAGCcAUuAGUcAGAUTsT 1179AD-14383  55% 11% cAcAAuGAuuuAAGGAcuGTsT 1180 cAGUCCUuAAAUcAUUGUGTsT1181 AD-14384  48%  9% ucuuuuucucGAuucAAAuTsT 1182AUUuGAAUCGAGAAAAAGATsT 1183 AD-14385  36%  2% cuuuuucucGAuucAAAucTsT1184 GAUUuGAAUCGAGAAAAAGTsT 1185 AD-14386  41%  7%AuuuucuGcucAcGAuGAGTsT 1186 CUcAUCGUGAGcAGAAAAUTsT 1187 AD-14387  38% 3% uuucuGcucAcGAuGAGuuTsT 1188 AACUcAUCGUGAGcAGAAATsT 1189 AD-14388 50%  4% AGAGcuAcAAAAccuAuccTsT 1190 GGAuAGGUUUUGuAGCUCUTsT 1191AD-14389  98%  6% GAGccAAAGGuAcAccAcuTsT 1192 AGUGGUGuACCUUUGGCUCTsT1193 AD-14390  43%  8% GccAAAGGuAcAccAcuAcTsT 1194GuAGUGGUGuACCUUUGGCTsT 1195 AD-14391  48%  4% GAAcuGuAcucuucucAGcTsT1196 GCUGAGAAGAGuAcAGUUCTsT 1197 AD-14392  44%  3%AGGuAAAuAucAccAAcAuTsT 1198 AUGUUGGUGAuAUUuACCUTsT 1199 AD-14393  37% 2% AGcuAcAAAAccuAuccuuTsT 1200 AAGGAuAGGUUUUGuAGCUTsT 1201 AD-14394114%  7% uGuGAAAGcAuuuAAuuccTsT 1202 GGAAUuAAAUGCUUUcAcATsT 1203AD-14395  55%  4% GcccAcuuuAGAGuAuAcATsT 1204 UGuAuACUCuAAAGUGGGCTsT1205 AD-14396  49%  5% uGuGccAcAcuccAAGAccTsT 1206GGUCUUGGAGUGUGGcAcATsT 1207 AD-14397  71%  6% AAAcuAAAuuGAucucGuATsT1208 uACGAGAUcAAUUuAGUUUTsT 1209 AD-14398  81%  7%uGAucucGuAGAAuuAucuTsT 1210 AGAuAAUUCuACGAGAUcATsT 1211 AD-14399  38% 4% GcGuGcAGucGGuccuccATsT 1212 UGGAGGACCGACUGcACGCTsT 1213 AD-14400106%  8% AAAGuuuAGAGAcAucuGATsT 1214 UcAGAUGUCUCuAAACUUUTsT 1215AD-14401  47%  3% cAGAAGGAAuAuGuAcAAATsT 1216 UUUGuAcAuAUUCCUUCUGTsT1217 AD-14402  31%  1% cGcccGAGAGuAccAGGGATsT 1218UCCCUGGuACUCUCGGGCGTsT 1219 AD-14403 105%  4% cGGAGGAGAuAGAAcGuuuTsT1220 AAACGUUCuAUCUCCUCCGTsT 1221 AD-14404   3%  1%AGAuAGAAcGuuuAAAAcGTsT 1222 CGUUUuAAACGUUCuAUCUTsT 1223 AD-14405  15% 1% GGAAcAGGAAcuucAcAAcTsT 1224 GUuGuGAAGUUCCuGUUCCTsT 1225 AD-14406 44%  5% GuGAGccAAAGGuAcAccATsT 1226 UGGUGuACCUUUGGCUcACTsT 1227AD-14407  41%  4% AuccucccuAGAcuucccuTsT 1228 AGGGAAGUCuAGGGAGGAUTsT1229 AD-14408 104%  3% cAcAcuccAAGAccuGuGcTsT 1230GcAcAGGUCUUGGAGUGUGTsT 1231 AD-14409  67%  4% AcAGAAGGAAuAuGuAcAATsT1232 UUGuAcAuAUUCCUUCUGUTsT 1233 AD-14410  22%  1%uuAGAGAcAucuGAcuuuGTsT 1234 cAAAGUcAGAUGUCUCuAATsT 1235 AD-14411  29% 3% AAuuGAucucGuAGAAuuATsT 1236 uAAUUCuACGAGAUcAAUUTsT 1237 AD-14412 31%  4%

dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2═-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioatelinkages were introduced by replacement of the iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referredto as -Chol-3′), an appropriately modified solid support was used forRNA synthesis. The modified solid support was prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole)was added and the mixture was stirred at room temperature untilcompletion of the reaction was ascertained by TLC. After 19 h thesolution was partitioned with dichloromethane (3×100 mL). The organiclayer was dried with anhydrous sodium sulfate, filtered and evaporated.The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It wasthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution was brought to room temperature and stirred further for 6 h.Completion of the reaction was ascertained by TLC. The reaction mixturewas concentrated under vacuum and ethyl acetate was added to precipitatediisopropyl urea. The suspension was filtered. The filtrate was washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer was dried over sodium sulfate and concentrated togive the crude product which was purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidinein dimethylformamide at 0° C. The solution was continued stirring for 1h. The reaction mixture was concentrated under vacuum, water was addedto the residue, and the product was extracted with ethyl acetate. Thecrude product was purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. Thesuspension was cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) wasadded. The reaction mixture was stirred overnight. The reaction mixturewas diluted with dichloromethane and washed with 10% hydrochloric acid.The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of drytoluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD was added slowly with stirring within 20 mins. Thetemperature was kept below 5° C. during the addition. The stirring wascontinued for 30 mins at 0° C. and 1 mL of glacial acetic acid wasadded, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water Theresultant mixture was extracted twice with 100 mL of dichloromethaneeach and the combined organic extracts were washed twice with 10 mL ofphosphate buffer each, dried, and evaporated to dryness. The residue wasdissolved in 60 mL of toluene, cooled to 0° C. and extracted with three50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extractswere adjusted to pH 3 with phosphoric acid, and extracted with five 40mL portions of chloroform which were combined, dried and evaporated todryness. The residue was purified by column chromatography using 25%ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) was added, the mixture was extracted with ethylacetate (3×40mL). The combined ethylacetate layer was dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which waspurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added withstirring. The reaction was carried out at room temperature overnight.The reaction was quenched by the addition of methanol. The reactionmixture was concentrated under vacuum and to the residue dichloromethane(50 mL) was added. The organic layer was washed with 1M aqueous sodiumbicarbonate. The organic layer was dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine was removed byevaporating with toluene. The crude product was purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)esterAH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture was dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and thesolution was stirred at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (40 mL) and washed with icecold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The residue was used as such for the next step.

Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM)was added. The suspension was agitated for 2 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The achieved loading of the CPG was measured bytaking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) was performed as described inWO 2004/065601, except that, for the cholesteryl derivative, theoxidation step was performed using the Beaucage reagent in order tointroduce a phosphorothioate linkage at the 5′-end of the nucleic acidoligomer.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 4.

TABLE 4 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation^(a) Nucleotide(s) A, a2′-deoxy-adenosine-5′-phosphate, adenosine-5′-phosphate C, c2′-deoxy-cytidine-5′-phosphate, cytidine-5′-phosphate G, g2′-deoxy-guanosine-5′-phosphate, guanosine-5′-phosphate T, t2′-deoxy-thymidine-5′-phosphate, thymidine-5′-phosphate U, u2′-deoxy-uridine-5′-phosphate, uridine-5′-phosphate N, n any2′-deoxy-nucleotide/nucleotide (G, A, C, or T, g, a, c or u) Am2′-O-methyladenosine-5′-phosphate Cm 2′-O-methylcytidine-5′-phosphate Gm2′-O-methylguanosine-5′-phosphate Tm 2′-O-methyl-thymidine-5′-phosphateUm 2′-O-methyluridine-5′-phosphate Af2′-fluoro-2′-deoxy-adenosine-5′-phosphate Cf2′-fluoro-2′-deoxy-cytidine-5′-phosphate Gf2′-fluoro-2′-deoxy-guanosine-5′-phosphate Tf2′-fluoro-2′-deoxy-thymidine-5′-phosphate Uf2′-fluoro-2′-deoxy-uridine-5′-phosphate A, C, G, T, U, underlined:nucleoside-5′-phosphorothioate a, c, g, t, u am, cm, gm, underlined:2-O-methyl-nucleoside-5′-phosphorothioate tm, um ^(a)capital lettersrepresent 2′-deoxyribonucleotides (DNA), lower case letters representribonucleotides (RNA)

dsRNA Expression Vectors

In another aspect of the invention, Eg5 specific dsRNA molecules thatmodulate Eg5 gene expression activity are expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be incorporated and inherited as a transgeneintegrated into the host genome. The transgene can also be constructedto permit it to be inherited as an extrachromosomal plasmid (Gassmann,et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. In apreferred embodiment, a dsRNA is expressed as an inverted repeat joinedby a linker polynucleotide sequence such that the dsRNA has a stem andloop structure.

The recombinant dsRNA expression vectors are generally DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector of the invention may be a eukaryotic RNA polymerase I (e.g.ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter oractin promoter or U1 snRNA promoter) or generally RNA polymerase IIIpromoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter,for example the T7 promoter, provided the expression plasmid alsoencodes T7 RNA polymerase required for transcription from a T7 promoter.The promoter can also direct transgene expression to the pancreas (see,e.g. the insulin regulatory sequence for pancreas (Bucchini et al.,1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would beable to choose the appropriate regulatory/promoter sequence based on theintended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g. Oligofectamine) ornon-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipidtransfections for dsRNA-mediated knockdowns targeting different regionsof a single Eg5 gene or multiple Eg5 genes over a period of a week ormore are also contemplated by the invention. Successful introduction ofthe vectors of the invention into host cells can be monitored usingvarious known methods. For example, transient transfection. can besignaled with a reporter, such as a fluorescent marker, such as GreenFluorescent Protein (GFP). Stable transfection. of ex vivo cells can beensured using markers that provide the transfected cell with resistanceto specific environmental factors (e.g., antibiotics and drugs), such ashygromycin B resistance.

The Eg5 specific dsRNA molecules can also be inserted into vectors andused as gene therapy vectors for human patients. Gene therapy vectorscan be delivered to a subject by, for example, intravenous injection,local administration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

Eg5 siRNA In Vitro Screening Via Cell Proliferation

As silencing of Eg5 has been shown to cause mitotic arrest (Weil, D, etal [2002] Biotechniques 33: 1244-8), a cell viability assay was used forsiRNA activity screening. HeLa cells (14000 per well [Screens 1 and 3]or 10000 per well [Screen2])) were seeded in 96-well plates andsimultaneously transfected with Lipofectamine 2000 (Invitrogen) at afinal siRNA concentration in the well of 30 nM and at finalconcentrations of 50 nM (1^(st) screen) and 25 nM (2^(nd) screen). Asubset of duplexes was tested at 25 nM in a third screen (Table 5).

Seventy-two hours post-transfection, cell proliferation was assayed theaddition of WST-1 reagent (Roche) to the culture medium, and subsequentabsorbance measurement at 450 nm. The absorbance value for control(non-transfected) cells was considered 100 percent, and absorbances forthe siRNA transfected wells were compared to the control value. Assayswere performed in sextuplicate for each of three screens. A subset ofthe siRNAs was further tested at a range of siRNA concentrations. Assayswere performed in HeLa cells (14000 per well; method same as above,Table 5).

TABLE 5 Relative absorbance at 450 nm Screen I Screen II Screen IIIDuplex mean sd Mean sd mean Sd AL-DP-6226 20 10 28 11 43 9 AL-DP-6227 6627 96 41 108 33 AL-DP-6228 56 28 76 22 78 18 AL-DP-6229 17 3 31 9 48 13AL-DP-6230 48 8 75 11 73 7 AL-DP-6231 8 1 21 4 41 10 AL-DP-6232 16 2 377 52 14 AL-DP-6233 31 9 37 6 49 12 AL-DP-6234 103 40 141 29 164 45AL-DP-6235 107 34 140 27 195 75 AL-DP-6236 48 12 54 12 56 12 AL-DP-623773 14 108 18 154 37 AL-DP-6238 64 9 103 10 105 24 AL-DP-6239 9 1 20 4 3111 AL-DP-6240 99 7 139 16 194 43 AL-DP-6241 43 9 54 12 66 19 AL-DP-62426 1 15 7 36 8 AL-DP-6243 7 2 19 5 33 13 AL-DP-6244 7 2 19 3 37 13AL-DP-6245 25 4 45 10 58 9 AL-DP-6246 34 8 65 10 66 13 AL-DP-6247 53 678 14 105 20 AL-DP-6248 7 0 22 7 39 12 AL-DP-6249 36 8 48 13 61 7

The nine siRNA duplexes that showed the greatest growth inhibition inTable 5 were re-tested at a range of siRNA concentrations in HeLa cells.The siRNA concentrations tested were 100 nM, 33.3 nM, 11.1 nM, 3.70 nM,1.23 nM, 0.41 nM, 0.14 nM and 0.046 nM. Assays were performed insextuplicate, and the concentration of each siRNA resulting in fiftypercent inhibition of cell proliferation (IC₅₀) was calculated. Thisdose-response analysis was performed between two and four times for eachduplex. Mean IC₅₀ values (nM) are given in Table 6.

TABLE 6 Duplex Mean IC50 AL-DP-6226 15.5 AL-DP-6229 3.4 AL-DP-6231 4.2AL-DP-6232 17.5 AL-DP-6239 4.4 AL-DP-6242 5.2 AL-DP-6243 2.6 AL-DP-62448.3 AL-DP-6248 1.9

Eg5 siRNA In Vitro Screening Via Cell Proliferation

Directly before transfection, Hela S3 (ATCC-Number: CCL-2.2, LCGPromochem GmbH, Wesel, Germany) cells were seeded at 1.5×10⁴ cells/wellon 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 75μl of growth medium (Ham's F12, 10% fetal calf serum, 100 upenicillin/100 μg/ml streptomycin, all from Biochrom AG, Berlin,Germany). Transfections were performed in quadruplicates. For each well0.5 μl Lipofectamine-2000 (Invitrogen GmbH, Karlsruhe, Germany) weremixed with 12 μl Opti-MEM (Invitrogen) and incubated for 15 min at roomtemperature. For the siRNA concentration being 50 nM in the 100 μltransfection volume, 1 μl of a 5 μM siRNA were mixed with 11.5 μlOpti-MEM per well, combined with the Lipofectamine-2000-Opti-MEM mixtureand again incubated for 15 minutes at room temperature.siRNA-Lipofectamine-2000-complexes were applied completely (25 μl eachper well) to the cells and cells were incubated for 24 h at 37° C. and5% CO₂ in a humidified incubator (Heraeus GmbH, Hanau). The single dosescreen was done once at 50 nM and at 25 nM, respectively.

Cells were harvested by applying 50 μl of lysis mixture (content of theQuantiGene bDNA-kit from Genospectra, Fremont, USA) to each wellcontaining 100 μl of growth medium and were lysed at 53° C. for 30 min.Afterwards, 50 μl of the lysates were incubated with probesets specificto human Eg5 and human GAPDH and proceeded according to themanufacturer's protocol for QuantiGene. In the end chemoluminescence wasmeasured in a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs(relative light units) and values obtained with the hEg5 probeset werenormalized to the respective GAPDH values for each well. Values obtainedwith siRNAs directed against Eg5 were related to the value obtained withan unspecific siRNA (directed against HCV) which was set to 100% (Tables1, 2 and 3).

Effective siRNAs from the screen were further characterized by doseresponse curves. Transfections of dose response curves were performed atthe following concentrations: 100 nM, 16.7 nM, 2.8 nM, 0.46 nM, 77picoM, 12.8 picoM, 2.1 picoM, 0.35 picoM, 59.5 fM, 9.9 fM and mock (nosiRNA) and diluted with Opti-MEM to a final concentration of 12.5 μlaccording to the above protocol. Data analysis was performed by usingthe Microsoft Excel add-in software XL-fit 4.2 (IDBS, Guildford, Surrey,UK) and applying the dose response model number 205 (Tables 1, 2 and 3).

The lead siRNA AD12115 was additionally analyzed by applying theWST-proliferation assay from Roche (as previously described).

A subset of 34 duplexes from Table 2 that showed greatest activity wasassayed by transfection in HeLa cells at final concentrations rangingfrom 100 nM to 10 fM. Transfections were performed in quadruplicate. Twodose-response assays were performed for each duplex. The concentrationgiving 20% (IC20), 50% (IC50) and 80% (IC80) reduction of KSP mRNA wascalculated for each duplex. (Table 7).

TABLE 7 Concentrations given in pM IC20s IC50s IC80s Duplex 2^(nd) 1st2nd 1st 2nd name 1^(st) screen screen screen screen screen screenAD12077 1.19 0.80 6.14 10.16 38.63 76.16 AD12078 25.43 25.43 156.18156.18 ND ND AD12085 9.08 1.24 40.57 8.52 257.68 81.26 AD12095 1.03 0.979.84 4.94 90.31 60.47 AD12113 4.00 5.94 17.18 28.14 490.83 441.30AD12115 0.60 0.41 3.79 3.39 23.45 23.45 AD12125 31.21 22.02 184.28166.15 896.85 1008.11 AD12134 2.59 5.51 17.87 22.00 116.36 107.03AD12149 0.72 0.50 4.51 3.91 30.29 40.89 AD12151 0.53 6.84 4.27 10.7222.88 43.01 AD12152 155.45 7.56 867.36 66.69 13165.27 ND AD12157 0.3026.23 14.60 92.08 14399.22 693.31 AD12166 0.20 0.93 3.71 3.86 46.2820.59 AD12180 28.85 28.85 101.06 101.06 847.21 847.21 AD12185 2.60 0.4215.55 13.91 109.80 120.63 AD12194 2.08 1.11 5.37 5.09 53.03 30.92AD12211 5.27 4.52 11.73 18.93 26.74 191.07 AD12257 4.56 5.20 21.68 22.75124.69 135.82 AD12280 2.37 4.53 6.89 20.23 64.80 104.82 AD12281 8.818.65 19.68 42.89 119.01 356.08 AD12282 7.71 456.42 20.09 558.00 ND NDAD12285 ND 1.28 57.30 7.31 261.79 42.53 AD12292 40.23 12.00 929.11109.10 ND ND AD12252 0.02 18.63 6.35 68.24 138.09 404.91 AD12275 25.7625.04 123.89 133.10 1054.54 776.25 AD12266 4.85 7.80 10.00 32.94 41.67162.65 AD12267 1.39 1.21 12.00 4.67 283.03 51.12 AD12264 0.92 2.07 8.5615.12 56.36 196.78 AD12268 2.29 3.67 22.16 25.64 258.27 150.84 AD122791.11 28.54 23.19 96.87 327.28 607.27 AD12256 7.20 33.52 46.49 138.04775.54 1076.76 AD12259 2.16 8.31 8.96 40.12 50.05 219.42 AD12276 19.496.14 89.60 59.60 672.51 736.72 AD12321 4.67 4.91 24.88 19.43 139.5089.49 (ND—not determined)

Silencing of Liver Eg5/KSP in Juvenile Rats Following Single-BolusAdministration of LNP01 Formulated siRNA

From birth until approximately 23 days of age, Eg5/KSP expression can bedetected in the growing rat liver. Target silencing with a formulatedEg5/KSP siRNA was evaluated in juvenile rats.

KSP Duplex Tested

Duplex ID Target Sense Antisense AD6248 Eg5/KS AccGAAGuGuuGuuuGuccTsGGAcAAAcAAcACUUCGGUTs P T T (SEQ ID NO: 1238) (SEQ ID NO: 1239)

Methods

Dosing of Animals.

Male, juvenile Sprague-Dawley rats (19 days old) were administeredsingle doses of lipidoid (“LNP01”) formulated siRNA via tail veininjection. Groups of ten animals received doses of 10 milligrams perkilogram (mg/kg) bodyweight of either AD6248 or an unspecific siRNA.Dose level refers to the amount of siRNA duplex administered in theformulation. A third group received phosphate-buffered saline. Animalswere sacrificed two days after siRNA administration. Livers weredissected, flash frozen in liquid Nitrogen and pulverized into powders.

mRNA Measurements.

Levels of Eg5/KSP mRNA were measured in livers from all treatmentgroups. Samples of each liver powder (approximately ten milligrams) werehomogenized in tissue lysis buffer containing proteinase K. Levels ofEg5/KSP and GAPDH mRNA were measured in triplicate for each sample usingthe Quantigene branched DNA assay (GenoSpectra). Mean values for Eg5/KSPwere normalized to mean GAPDH values for each sample. Group means weredetermined and normalized to the PBS group for each experiment.

Statistical Analysis.

Significance was determined by ANOVA followed by the Tukey post-hoc test

Results

Data Summary

Mean values (±standard deviation) for Eg5/KSP mRNA are given.Statistical significance (p value) versus the PBS group is shown (ns,not significant [p>0.05]).

Experiment 1

VEGF/GAPDH p value PBS 1.0 ± 0.47 AD6248 10 mg/kg 0.47 ± 0.12  <0.001unspec 10 mg/kg 1.0 ± 0.26 ns

A statistically significant reduction in liver Eg5/KSP mRNA was obtainedfollowing treatment with formulated AD6248 at a dose of 10 mg/kg.

Silencing of Rat Liver VEGF Following Intravenous Infusion of LNP01Formulated siRNA Duplexes

A “lipidoid” formulation comprising an equimolar mixture of two siRNAswas administered to rats. One siRNA (AD3133) was directed towards VEGF.The other (AD12115) was directed towards Eg5/KSP. Since Eg5/KSPexpression is nearly undetectable in the adult rat liver, only VEGFlevels were measured following siRNA treatment.

siRNA Duplexes Administered

Duplex ID Target Sense Antisense AD12115 Eg5/KSP ucGAGAAucuAAAcuAAcuTsTAGUuAGUUuAGAUUCUCGATsT (SEQ ID NO: 1240) (SEQ ID NO: 1241) AD3133 VEGFGcAcAuAGGAGAGAuGAGCUsU AAGCUcAUCUCUCCuAuGuGCusG (SEQ ID NO: 1242)(SEQ ID NO: 1243) Key: A,G , C, U-ribonucleotides; c,u-2′-O-Meribonucleotides; s-phorphorothioate.

Methods

Dosing of Animals.

Adult, female Sprague-Dawley rats were administered lipidoid (“LNP01”)formulated siRNA by a two-hour infusion into the femoral vein. Groups offour animals received doses of 5, 10 and 15 milligrams per kilogram(mg/kg) bodyweight of formulated siRNA. Dose level refers to the totalamount of siRNA duplex administered in the formulation. A fourth groupreceived phosphate-buffered saline. Animals were sacrificed 72 hoursafter the end of the siRNA infusion. Livers were dissected, flash frozenin liquid Nitrogen and pulverized into powders.

Formulation Procedure

The lipidoid ND98.4HCl (MW 1487) (Formula 1), Cholesterol(Sigma-Aldrich), and PEG-Ceramide C 16 (Avanti Polar Lipids) were usedto prepare lipid-siRNA nanoparticles. Stock solutions of each in ethanolwere prepared: ND98, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16,100 mg/mL. ND98, Cholesterol, and PEG-Ceramide C16 stock solutions werethen combined in a 42:48:10 molar ratio. Combined lipid solution wasmixed rapidly with aqueous siRNA (in sodium acetate pH 5) such that thefinal ethanol concentration was 35-45% and the final sodium acetateconcentration was 100-300 mM. Lipid-siRNA nanoparticles formedspontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture was in some casesextruded through a polycarbonate membrane (100 nm cut-off) using athermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In othercases, the extrusion step was omitted. Ethanol removal and simultaneousbuffer exchange was accomplished by either dialysis or tangential flowfiltration. Buffer was exchanged to phosphate buffered saline (PBS) pH7.2.

Characterization of Formulations

Formulations prepared by either the standard or extrusion-free methodare characterized in a similar manner. Formulations are firstcharacterized by visual inspection. They should be whitish translucentsolutions free from aggregates or sediment. Particle size and particlesize distribution of lipid-nanoparticles are measured by dynamic lightscattering using a Malvern Zetasizer Nano Z S (Malvern, USA). Particlesshould be 20-300 nm, and ideally, 40-100 nm in size. The particle sizedistribution should be unimodal. The total siRNA concentration in theformulation, as well as the entrapped fraction, is estimated using a dyeexclusion assay. A sample of the formulated siRNA is incubated with theRNA-binding dye Ribogreen (Molecular Probes) in the presence or absenceof a formulation disrupting surfactant, 0.5% Triton-X100. The totalsiRNA in the formulation is determined by the signal from the samplecontaining the surfactant, relative to a standard curve. The entrappedfraction is determined by subtracting the “free” siRNA content (asmeasured by the signal in the absence of surfactant) from the totalsiRNA content. Percent entrapped siRNA is typically >85%.

mRNA Measurements.

Samples of each liver powder (approximately ten milligrams) werehomogenized in tissue lysis buffer containing proteinase K. Levels ofVEGF and GAPDH mRNA were measured in triplicate for each sample usingthe Quantigene branched DNA assay (GenoSpectra). Mean values for VEGFwere normalized to mean GAPDH values for each sample. Group means weredetermined and normalized to the PBS group for each experiment.

Protein Measurements.

Samples of each liver powder (approximately 60 milligrams) werehomogenized in 1 ml RIPA buffer. Total protein concentrations weredetermined using the Micro BCA protein assay kit (Pierce). Samples oftotal protein from each animal was used to determine VEGF protein levelsusing a VEGF ELISA assay (R&D systems). Group means were determined andnormalized to the PBS group for each experiment.

Statistical Analysis.

Significance was determined by ANOVA followed by the Tukey post-hoc test

Results

Data Summary

Mean values (±standard deviation) for mRNA (VEGF/GAPDH) and protein(rel. VEGF) are shown for each treatment group. Statistical significance(p value) versus the PBS group for each experiment is shown.

VEGF/GAPDH p value rel VEGF p value PBS  1.0 ± 0.17  1.0 ± 0.17  5 mg/kg0.74 ± 0.12 <0.05 0.23 ± 0.03 <0.001 10 mg/kg 0.65 ± 0.12 <0.005 0.22 ±0.03 <0.001 15 mg/kg 0.49 ± 0.17 <0.001 0.20 ± 0.04 <0.001

Statistically significant reductions in liver VEGF mRNA and protein weremeasured at all three siRNA dose levels.

We claim:
 1. A composition comprising a double-stranded ribonucleic acid(dsRNA) for inhibiting expression of a human kinesin family member 11(Eg5) gene in a cell, wherein the dsRNA comprises a first sense strandcomprising a first sequence and a first antisense strand comprising asecond sequence complementary to an Eg5 mRNA, wherein the first sequenceis complementary to the second sequence and wherein the dsRNA is between15 and 30 base pairs in length.
 2. The composition of claim 1, whereinthe dsRNA comprises at least one modified nucleotide.
 3. The compositionof claim 2, wherein the modified nucleotide is chosen from the group of:a 2′-O-methyl modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group.
 4. Thecomposition of claim 2, wherein the modified nucleotide is chosen fromthe group of: a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholinonucleotide, a phosphoramidate, and a non-natural base comprisingnucleotide.
 5. The composition of claim 2, wherein the first dsRNAcomprises at least one 2′-O-methyl modified ribonucleotide and at leastone phosphorothioate.
 6. The composition of claim 1, wherein thecomposition, upon contact with a cell expressing Eg5, inhibitsexpression of Eg5 gene by at least 40%.
 7. The composition of claim 1,wherein the dsRNA is 19-21 base pairs in length.
 8. An isolated cellcomprising the composition of claim
 1. 9. A vector comprising aregulatory sequence operably linked to a nucleotide sequence thatencodes at least one strand of the dsRNA of the composition of claim 1.10. An isolated cell comprising the vector of claim
 9. 11. Apharmaceutical composition for inhibiting Eg5 gene expression comprisingthe composition of claim 1 and a pharmaceutically acceptable carrier.12. A method for inhibiting Eg5 gene expression in a cell, the methodcomprising: introducing into the cell the composition of claim 1; andmaintaining the cell for a time sufficient to obtain degradation of themRNA transcript of the Eg5 gene, thereby inhibiting expression of theEg5 gene in the cell.
 13. A method of treating or managing pathologicalprocesses mediated by human Eg5 expression comprising administering to apatient in need of such treatment or management a therapeuticallyeffective amount of the composition of claim 1.